Multianalyte assay

ABSTRACT

The invention provides compositions, systems and methods for detecting multiple analytes from a sample.

CROSS-REFERENCE

This present application is a divisional of U.S. application Ser. No.12/596,835 filed on May 17, 2010, which is a national phase stageapplication of PCT/US2008/062088 filed Apr. 30, 2008, which claimsbenefit of priority to U.S. Provisional Application Ser. No. 60/915,051,which was filed Apr. 30, 2007. The content of these related applicationsis incorporated herein in its entirety. This application is also relatedto U.S. patent application Ser. No. 11/677,559, filed Feb. 21, 2007,which is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Contract number 200-2007-19345 Center for Disease Control.

BACKGROUND OF THE INVENTION

While analyte detection device and assays are known in the art, such asthose used for diagnostics for infectious diseases, they lack sufficientsensitivity and specificity to detect multiple analytes using a singleassay device. A new generation of multianalyte assay devices that areintended for rapid detection of multiple analytes require improvedsensitivity and specificity. Therefore, there is a need to developdevices and methods to effectively detect a plurality of analytes andthat are capable of minimizing false positive and false negativeresults, and to often screen samples that contain a mixed pool of targetand non-target analytes present in a sample.

SUMMARY OF THE INVENTION

In various aspects of the invention, systems, devices and methods areprovided for detection of multiple analytes present in a sample. In oneaspect of the invention, devices and processes are provided to provideenhanced sensitivity and specificity to detect a plurality of differentanalytes in a sample.

In one aspect of the invention, a kit is provided comprising a SampleCollection Device (SCD) and Test Device (TD) configured to providedenhanced sensitivity and specificity for detecting one or more analytepresent in a sample. Such samples can contain a mixed pool of non-targetand target analytes.

In another aspect of the invention a system is provided for detectingone or more analytes present in a sample which minimizes the stepsrequired to process a sample and simplifies the process for reading aresult. Furthermore, the system reduces the level of training requiredfor an operator to process and read results. In one embodiment, thesystem comprises a SCD, TD and reader.

In yet another aspect methods are provided for detecting one or moreanalytes present in a sample, comprising administering a sample todevices and systems of the invention, which are configured to providedenhanced detection sensitivity and specificity. In various embodiments,samples screened are from any source and the one or more analytesdetected are any entity that is detectable using a detectable entitythat binds to an analyte. Analytes include but are not limited to virusor virus components, bacteria or bacterial components, mammalian cell ormammalian cell components, and non-mammalian cell or non-mammalian cellcomponents. For example, in some embodiments the invention providesdetection of Influenza types A and B as well as Subtypes of Type A, H1and H3 and H5.

In a further aspect of the invention, reagents, buffers and immunoassaycomponents are provided, as described herein, which enhance thespecificity and sensitivity for detection of multiple analytes ifpresent in the same sample. Such reagents, buffers and immunoassaycomponents include but are not limited to detection probes, captureprobes, extraction buffers, antibodies and moieties.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to file same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates an embodiment of sample collection device (SCD).

FIG. 2 illustrates a test device (“TD”) designed for ease of use,barcode compatible and reducing/eliminating the potential of forming anaerosol of the specimen by having the Sample Collection Device (“SCD”)attach directly to the TD.

FIG. 3 illustrates an optical reader.

FIG. 4 illustrates one example for detection of analytes with greatersensitivity (−2 logs improvement over prior art) and specificity.

FIGS. 5A and 5B depict one embodiment of the test device; FIG. 5A showsthe component of the test device; FIG. 5B shows the role of pRNA in thetest device.

FIG. 6 illustrate one embodiment for detection of an analyte.

FIG. 7, panels A, B and C, illustrate one embodiment of work flow forprocessing a sample using a detection system.

FIG. 8 illustrates a strip/dipstick example for detection influenza.

FIG. 9 illustrates a NT-proBNP standard curve.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. Additionalembodiments for compositions, methods or systems disclosed herein aredisclosed in related U.S. patent application Ser. No. 11/677,559.

In one aspect of the invention systems, devices and methods provide arapid, sensitive process for detecting multiple analytes in a biologicalsample with high sensitivity and specificity. For example, in someembodiments, an assay is provided which has high sensitivity and fewerfalse positives than conventional assays. A further embodiment is toprovide an apparatus or system for detection of low levels of analytespresent in biological samples. In yet another embodiment, an assaysystem is provided which involves a minimal number of procedural steps,and yields reliable results even when used by untrained persons.

In one embodiment, a system is provided for testing a sample for thepresence of a plurality of analytes. Furthermore, detection is in amatter of minutes. In additional embodiments, the invention provideresults which are specific and sensitive for one or more targetanalytes, notwithstanding that results can be read one to several hoursafter completion of a reaction necessary to obtain a result.

As used herein the term “analyte” or “analytes” refers to the compoundor composition to be detected or measured and which has at least oneepitope or binding site. The analyte can be any substance for whichthere exists a naturally occurring analyte specific binding member orfor which an analyte-specific binding member can be prepared. e.g.,carbohydrate and lectin, hormone and receptor, complementary nucleicacids, and the like. Further, possible analytes include virtually anycompound, composition, aggregation, or other substance which may beimmunologically detected. That is, the analyte, or portion thereof, willbe antigenic or haptenic having at least one determinant site, or willbe a member of a naturally occurring binding pair. In other embodiments,one or more analyte detected is an antibody (e.g., IgG, IgM) in a sample(e.g., urine, oral fluid, blood, plasma or serum sample) where theantibody is specific for a virus or virus component, bacteria orbacteria component, cancer cell or tumor antigen. For example, bydetecting one or more antibody, the assay indicates that the patient hasbeen previously infected by an infectious agent or suffers an underlyingcondition with which the antibody is associated. In further embodiments,allergy detection testing comprises detecting the presence of specificIgG, IgM and/or IgE Ab in a subjects oral fluid, whole blood, urine,plasma or serum to specific allergens.

Analytes include, but are not limited to, toxins, organic compounds,proteins, peptides, microorganisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances. The termanalyte also includes any antigenic substances, haptens, antibodies,macromolecules, and combinations thereof. A non-exhaustive list ofexemplary analytes is set forth in U.S. Pat. No. 4,366,241, at column19, line 7 through column 26, line 42; U.S. Pat. Nos. 4,299,916;4,275,149; and 4,806,311. Therefore, in various embodiments, one or moreanalyte is detected from a sample obtained from a subject.

Samples

A sample is any material to be tested for the presence and/orconcentration of an analyte. In general, a biological sample can be anysample taken from a subject, e.g., non-human animal or human andutilized in the test devices. For example, a biological sample can be asample of any body fluid, cells, or tissue samples from a biopsy. Bodyfluid samples can include without any limitation blood, urine, sputum,semen, feces, saliva, bile, cerebral fluid, nasal swab, urogenital swab,nasal aspirate, spinal fluid, etc. Biological samples can also includeany sample derived from a sample taken directly from a subject, e.g.,human. For example, a biological sample can be the plasma fraction of ablood sample, serum, protein or nucleic acid extraction of the collectedcells or tissues or from a specimen that has been treated in a way toimprove the detectability of the specimen, for example, a lysis buffercontaining a mucolytic agent that breaks down the mucens in a nasalspecimen significantly reducing the viscosity of the specimen and adetergent to lyse the virus thereby releasing antigens and making themavailable for detection by the assay. A sample can be from any subjectanimal, including but not limited to, human, bird, porcine, equine,bovine, murine, cat, dog or sheep.

For example, a sample can be derived from any source, such as aphysiological fluid, including blood, serum, plasma, saliva or oralfluid, sputum, ocular lens fluid, nasal fluid, nasopharyngeal or nasalpharyngeal swab or aspirate, sweat, urine, milk, ascites fluid, mucous,synovial fluid, peritoneal fluid, transdermal exudates, pharyngealexudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinalfluid, semen, cervical mucus, vaginal or urethral secretions, amnioticfluid, and the like. Herein, fluid homogenates of cellular tissues suchas, for example, hair, skin and nail scrapings and meat extracts arealso considered biological fluids. Pretreatment may involve preparingplasma from blood, diluting or treating viscous fluids, and the like.Methods of treatment can involve filtration, distillation, separation,concentration, inactivation of interfering components, and the additionof reagents. Besides physiological fluids, other samples can be usedsuch as water, food products, soil extracts, and the like for theperformance of industrial, environmental, or food production assays aswell as diagnostic assays. In addition, a solid material suspected ofcontaining the analyte can be used as the test sample once it ismodified to form a liquid medium or to release the analyte. Theselection and pretreatment of biological, industrial, and environmentalsamples prior to testing is well known in the art and need not bedescribed further.

Other fields of interest include the diagnosis of veterinary diseases,analysis of meat, poultry, fish for bacterial contamination, inspectionof food plants, food grains, fruit, dairy products (processed orunprocessed), restaurants, hospitals and other public facilities,analysis of environmental samples including water for beach, ocean,lakes or swimming pool contamination. Analytes detected by these testsinclude viral and bacterial antigens as well as chemicals including, forexample, lead, pesticides, hormones, drugs and their metabolites,hydrocarbons and all kinds of organic or inorganic compounds.

In one aspect of the invention a kit is provided for detection of one ormore analytes comprising: a sample collection device comprising; asample tube 10; a sample collection implement 13; a dropper cap 14; acompartment comprising an extraction buffer FIG. 7, panel A; and a testdevice 19, FIG. 7, panel B comprising one or more addressable regionsconfigured for detection of one or more different analytes.

In some embodiments, the SCD comprises an extraction buffer comprisingfrom about 0.75 to about 1.125M of salt in a buffered solution. In oneembodiment, a salt in buffered solution is about 0.75M, 1M, 1.1M or1.125M. In further embodiments, the extraction buffer contains about1.0% to about 1.5% saponin. In yet further embodiments, saponin is at aconcentration of about 0.75%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%,1.7%, 1.8%, 1.9% or 2.0%. In further embodiments, a zwitterionic agent(e.g., Zwittergent 3/12) is provided to enhance extract of one or moreanalyte. For example, a zwitterion agent is provided in an extractionbuffer at about 0.1% to about 1.5%. In yet further embodiments, aZwittergent agent is at a concentration of about 0.1%, 0.15%, 0.175%,0.2%, 0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.350%, 0.375%, 0.4%, 0.425%,0.450%, 0.475%, 0.5%, 0.525%, 0.550%, 0.575%, 0.6%, 0.7%, 0.75%, 1.0%,1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0%. Examplesof zwitterionic agent include Zwittergent 3/12; most amino acids atphysiological pH, Used as buffering agents in Good's buffers: theamino-sulfonic acid based MES, MOPS, HEPES, PIPES or CAPS; theaminocarboxylic acid (amino acid) based glycine, its derivatives bicineand tricine, and alanine; CHAPSO; natural products like the alkaloidspsilocybin and lysergic acid; betaines; Quinonoid zwitterions; drugssuch as Fexofenadine (Allegra) and Cephaloridine; 2-(NMorpholino)ethanesulfonic acid, (3-[N-Morpholinojjpropanesulfonic acid,2-[(2-Amino-2-oxoethyl)amino]ethane sulfonic acid,Piperazine-N,N′-bis(2-ethane sulfonic acid),3-(N-Morpholino)-2-hydroxypropanesulfonic acid,N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,3-(N-Morpholino)propanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethane sulfonic acid), N-Tris(hydroxymethyl)methyl-2aminoethanesulfonic acid,3-[N,N-Bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid,3-(N-Tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid,N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid),Piperazine-N,N′-bis(2-hydroxypropanesulfonic acid),N-(2-Hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), N-Tris(hydroxyrnethyl)methyl-3-aminopropanesulfonic acid, 3-[(1,I-)Dimethy1-2-hydroxyethyl)amino J-2-hydroxypropanesulfonic, acid,2-(N-Cyclohexylamino) ethanesulfonic acid,3-{cyclohexylamino)-2-hydroxy-lpropanesulfonic acid,2-Amino-2-methyl-1-propanol, 3-(cyclohexylamino)-1-propanesulfonic acid,or mixtures thereof

For example, extraction agents of the invention enhance extraction ofmembrane antigens including but not limited to haemagglutinin.Haemagglutinins are membrane glycoproteins well known to be associatedwith sphingolipid/cholesterol-enriched membrane domains (i.e. rafts;Detergent-insoluble glycolipid-enriched complexes (DIGs); or detergentresistant membranes, (DRMs>>. In one embodiment, the combination of azwitterionic detergent (e.g., Zwittergent 3-12), with Saponin(containing 20-35% sapogenin) mediates a rapid solubilization of HI, H3and H5 influenza haemagglutinins. The solubilization by these agentsenhance assay sensitivity for the haemagglutnin antigens withoutinterfering with the detection of the nucleoproteins from Influenza A orInfluenza B in the same assay.

In one embodiment the extraction reagent (buffer) is contained in a bulbin the handle of a SCD and a mucolytic agent is placed in the distal endof the SCD in dried or lyophilized state in such a way that when it isexposed to the extraction reagent the mucolytic reagent will be releasedand will aid in extracting the sample found on a swab or in a liquidspecimen.

In some aspects of the invention, one or more analytes detected includebut are not limited to one or more virus or virus components, one ormore bacteria or bacterial components, one or more cancer cell or cancercell component, one or more analyte associated with a heart damage,disease or condition, one or more analyte associated with a braindamage, disease or condition. In some embodiments, the one or more virusor viral components detected, are influenza A and/or influenza B. Infurther embodiments, the influenza A includes subtypes of a formulaHxNy, where x is 1 through 16, and y is 1 through 9, or any combinationof xy thereof. In one embodiment, the influenza A is H5N1.

In one embodiment, the dropper cap 14 comprises a plurality of detectionprobes and plurality of capture probes. In further embodiments, thedropper can comprise additional reagents or buffers (e.g., mucolyticreagent, salts, detergents, etc.). As used herein, a “capture probe”refers to a binding agent linked to a capture moiety and a “detectionprobe” or “label probe” refers to a binding agent linked to a label,detectable moiety or signal producing moiety, wherein each of thecapture probe and detection probe is capable of specifically binding toa target analyte. Furthermore, for clarity, a “capture moiety partner”in the context of the Test Device (described below) refers to acomplement, cognate or partner molecule that specifically binds to acapture moiety comprised on a capture probe. In various embodiments,devices and methods of the invention are configured to detect 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 different target analytes.

In one aspect of the invention, devices, systems and methods of theinvention are configured to detect one or more analytes at a sensitivityand/or specificity of about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100%. In some embodiments, devices, systems and methods ofthe invention are configured to provide a limit of sensitivity andspecificity for detection an analyte of at least about 97%.

In yet other embodiments, devices, systems and methods of the inventionare configured to detect of one or more analyte present where detectionis at a sensitivity of at about (in pg/mL) 0.010, 0.020, 0.030, 0.040,0.050, 0.060, 0.070, 0.080, 0.090, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60,0.70, 0.80, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0,12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0,40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100, 150, 200, 250, 300, 400or 500 pg/ml. In one embodiment, the sensitivity is at least from about0.010 to about 0.10 pg/mL. In one embodiment, the sensitivity is atleast about 0.030 pg/mL.

In some embodiments, detection of one or more analyte is at asensitivity of about 0.25, 0.50, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5,3.0, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 80, 90 about 100 attamoles. In one embodiment, thesensitivity for detection of each of one or more analytes is least about1.5 attamoles.

In some embodiments, devices, systems and methods of the invention areconfigured to provide a limit of sensitivity for detection of each ofsaid different analyte as measured by tissue culture infectious dose 50(TCID₅₀) is from about TCID₅₀ of 1, 5, 10, 15, 20, 25, 50, 75, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950 or about 1000 per mL. In oneembodiment, sensitivity for detection of an analyte is at about 10 toabout 200 TCID₅₀ per mL of sample. In one embodiment, sensitivity is atabout 10 TCID₅₀.

In another aspect of the invention a method is provided for assaying asample to detect one or more analytes comprising: administering a sampleto a sample collection device (SCD) comprising; at least pair of adetection probe and a capture probe, each of which is capable ofspecifically binding an analyte; and a buffer for extracting saidanalyte from said sample; administering a sample from said SCD to a testdevice (TD) configured to detect said one or more analytes, wherein saidTD comprises; a aperture for receiving said SCD and where said TD doesnot comprise a mobilizable entity capable of specifically binding saidone or more analyte; at least one immobilized capture moiety which iscapable of specifically binding a component of said capture probe; andthereby assaying said sample to detect said one or more analytes.

In various embodiments, the method comprises contacting a sample with anextraction buffer contains a salt in a buffered solution inconcentrations as disclosed herein. In further embodiments, anextraction buffer contains saponin in concentrations as disclosed herein(e.g., supra).

In further embodiments, one or more analytes detected are one or morevirus or virus components, one or more bacteria or bacterial components,one or more cancer cell or cancer cell component, one or more analyteassociated with a heart damage, disease or condition or one or moreanalyte associated with a brain damage, disease or condition.

In one embodiment, the one or more analytes detected comprise influenzaA and/or influenza B or components thereof. In further embodiments, theinfluenza A includes subtypes of a formula HxNy, where x is 1 through16, and y is 1 through 9, or any combination of xy thereof. In oneembodiment, the influenza A includes H5N1.

In another aspect of the invention a system is provided comprising: asample collection device comprising, one or more pairs of detectionprobe and capture probe, wherein said one or more pairs is configured todetect one or more different analyte, a sampling implement; a testdevice comprising one or more immobilized capture moieties which arecapable of specifically binding to said one or more capture probe, withthe proviso that said test device does not comprise a mobilizablebinding agent which is capable of binding specifically to said one ormore different analyte.

In further embodiments, the system comprises a buffer containing salt ina buffered solution at concentrations disclosed herein. In furtherembodiments, the buffer comprises saponin at concentrations providedherein. Reagents for extraction of sample can be comprised in a SCD ofthe invention or mixed with a sample to form a solution that issubsequently added to a SCD of the invention.

In various embodiments, the system is configured for detection of one ormore analytes including but not limited to one or more virus or viruscomponents, one or more bacteria or bacterial components, one or morecancer cell or cancer cell component, one or more analyte associatedwith a heart damage, disease or condition or one or more analyteassociated with a brain damage, disease or condition.

In some embodiments, the system is configured to detect one or moreanalytes comprising influenza A and/or influenza B virus or componentsthereof. In further embodiments, the influenza A includes subtypes of aformula HxNy, where x is 1 through 16, and y is 1 through 9, or anycombination of xy thereof. In one embodiment, the influenza A includesH5N1.

In various embodiments, the system is configured to provide detection ofone or more analytes at sensitivity and specificity as disclosed herein.For example, in one embodiment, sensitivity and/or specificity is atleast about 5 97%.

In another embodiment, a system of the invention is configured toprovide detection of one or more analyte at a sensitivity of at leastabout 0.030 pg/ml. In another embodiment, the system is configured toprovide a limit of sensitivity for detection of each of said differentanalyte of at least about 1.5 attamoles. In yet another embodiment, thesystem is configured to provide sensitivity of at least about 10 toabout 200 TCID₅₀ per mL for detection of an analyte as measured bytissue culture infectious dose 50 (TCID₅₀). In one embodiment,sensitivity is at least about TCID₅₀.

In another aspect of the invention a method is provided for detectingone or more analytes comprising, administering a sample to a testdevice, wherein a device is configured to detect each of said one ormore peptide at a sensitivity of at least about 0.30 pg/ml or 1.7attamoles (10⁻¹⁸), and of at least about 97% relative to a non-targetanalyte. In a further embodiment, one or more analyte (e.g.,polypeptide, peptide, nucleic acid) is detected, which is associatedwith a condition selected from a group consisting of a brain condition,damage or disease, a heart condition, damage or disease, a cancer orneoplastic condition or disease, a liver condition, damage or disease, akidney condition, damage or disease, or a combination thereof. In someembodiments, the one or more analyte detected is BNP, NT-proBNP, proBNP,CNP, and ANP.

In another embodiment, a method is provided for detection at least twoor more virus in a sample comprising, administering a sample to a systemcomprising a SCD and TD, wherein said system is configured to detecteach of said two or more virus at a sensitivity of about and specificityof at least about 97%.

In one embodiment, a method is provided for detection of one or moreanalyte comprising BNP, NTproBNP, proBNP, CNP, and ANP, wherein saidmethod comprises administering a sample to a device configured to detectthe one or more analyte at a sensitivity of from about 5 to 10 pg/rnL.

In various embodiments, a SCD (e.g., FIG. 1, 10) comprises a samplingimplement that provides a means to collect a sample 21 from a subject,wherein the sampling implement is in fluid communication to the upperchamber via a sampling implement holder. The sampling implement isdisposed at the distal end of a shaft, which shaft can be solid, hollowor semi-permeable. In some embodiments, the sampling implement is aswab, a comb, a brush, a spatula, a rod, a foam, a flocculated substrateor a spun substrate. In other embodiments, a sample can be added to theSCD 10 where the sample is a stored or previously obtained sample (e.g.,archived blood sample). Therefore, in one embodiment a SCD can also beutilized without the need for a sample collection implement 21.

In various embodiments, an SCD comprises one or more sealed upperchambers wherein the seal functions as a valve to control fluidcommunication between the upper chamber and lower chamber of an SCD. Insome embodiments, the valve can be a break-away valve, a flapper valve,a twist, screw, rupturable, puncturable or breakable valve. In otherembodiments, the upper chamber can contain one or more ampoules whichprevent solutions contained therein to flow to the lower chamber, unlesspressure is exerted to rupture, puncture or break the ampoule so as torelease any contents therein.

One aspect of the invention is directed to a SCD comprising a samplereservoir upstream of a plunger implement, a plurality of sealableapertures for delivery of one or more solutions, a substrate forfiltering one or more compounds from a sample administered to the samplereservoir and reagents that are capable of specifically binding at leastone analyte in said sample.

In another aspect of the invention, a Test Device is provided fordetection of one or more analytes, wherein the device comprises alateral flow membrane in a body, a chamber upstream of the lateral flowmembrane containing a fluid or solution, wherein a gap is disposedbetween said chamber and said lateral flow membrane thus precludingfluid communication between the chamber and the lateral flow membrane.In one embodiment, the pressure exerted on the chamber pushes close thegap thus forming fluid communication between the chamber and the lateralflow membrane. In one embodiment, an opening into which a distal end ofan SCD fits, is disposed directly above a wicking pad that is disposeddownstream of the gap, but upstream of the lateral flow membrane.

In one embodiment, the Test Device chamber comprises one or moresubchambers containing the same or different solutions. In otherembodiments, the chamber of subchambers comprise one or more ampoules orpackets that are breakable, puncurable or rupturable. Thus, wherepressure is exerted on such ampoules or packets the contents arecontrollably released. As described herein, a Test Device can comprise agap means or not comprise a gap means for disrupting fluid communicationfrom the chamber to the lateral flow membrane. A Test Device gap can befrom zero to 3.0, 0.5, to 3.5, 1.0 to 2.5, 1.0 to 3.0, or 2.0 to 4.0 mm.

In some embodiments, a Test Device can comprise a body housing thelateral flow membrane, wherein the body provides one or a plurality ofwindows through which the lateral flow membrane is visible. In variousembodiments described herein, a Test Device comprises a lateral flowmembrane that comprises a wicking substrate and an absorbent substrateupstream or downstream of the test zones disposed on said lateral flowmembrane. In some embodiments, a substrate for collecting a small volumeof sample for archiving is provided in a SCD or Test Device. In oneembodiment, the substrate providing such archiving means is a filter,membrane or paper that collects a small volume of sample and saidsubstrate is subsequently removed from the device.

In various embodiments, an SCD and/or Test Device comprises one or moreidentical identifiable tags, which can be removed from one device andplaced on another device.

In some embodiments, the Test Device is shaped to only fit (specializedadaptor shape) into the receiving port of a reader if the upstreamchamber has been depressed thus indicating that wash buffer or chasebuffer contained therein has been released through the lateral flowmembrane. In such embodiments, the Test Device and Reader specializedadaptor provides a means to verify that chase buffer or solution in theupstream chamber of the Test Device has been released and thus washedany sample present upstream of the lateral flow membrane through thelateral flow membrane. Thereby, the specialized adaptor provides a“safety means” to prevent reading of unprocessed samples.

In another aspect of the invention, the processed samples are runthrough the Test Device's lateral flow membrane, but can be placed asidefrom 30 minutes to several hours. In various embodiments, a plurality ofsamples can be run through the Test Device but read at 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11 or 12 hours later, with consistent and accuratesignals.

In certain aspects of the invention, the devices disclosed herein areutilized in methods for detection of one or more analyte that may bepresent in a sample. In some embodiments, methods are directed todetecting one or more strain of an infectious agent. In one embodiment,a method is provided to detect one or more influenza virus and subtypesthereof. For example, methods are provided for detection of influenzavirus A and B, and subtype of influenza A that may be present in asingle sample.

In certain aspects of the invention a sample collection device (SCD) isprovided for detection of one or more target antigens or analytes thatmay be present in a sample. The sample collection device can be utilizedin conjunction with a test device (e.g., FIG. 1). In one aspect, asystem is provided for detection of one or more analyte comprising aSCD, a Test Device and a Reader, as further described herein (e.g., FIG.7).

In various embodiments, a SCD comprises one or more upper sealedchambers, which can contain the same or different solutions. In oneembodiment, the upper sealed chamber comprises at least two compartmentsor subchambers each comprising the same or different reagents and/orbuffers. In other embodiments, the upper chamber can comprisepuncturable, breakable or rupturable ampoules. In some embodiments, aSCD provides the necessary reagents to form a complex with one or moredifferent target antigens that may be present in a sample, wherein thecomplex comprises a capture moiety and a detectable label, a Test Deviceprovides the necessary means to addressably capture one or morecomplexes so formed and a Reader which provides a means to detect one ormore signals from addressably captured complexes.

In various embodiments, an upper sealed chamber comprising extractionbuffer and/or reagents, a sample collection implement, a samplecollection implement holder 10 and a plurality of reagents, wherein thereagents comprise a plurality of specific binding pairs, where each paircomprises a label 51 conjugated to first specific binding agent 52, 53(“detection probe” or “label probe”) and a capture moiety 56, 57conjugated to a second specific binding agent 60 (“capture probe”) wherethe first and second specific binding agents specifically bind a targetantigen to form a complex. The capture moiety can be “captured” to apartner capture moiety 58, 59 immobilized on a substrate binds to thecapture moiety-specific binding agent conjugate. In various embodiments,the specific binding agents are antibodies, thus a specific binding paircomprises an antibody-label conjugate (“label probe” or “detectionprobe”) or antibody-capture moiety (“capture probe”). In suchembodiments, a “partner capture moiety” is comprised on a test membranedisposed in a Test Device, which partner binds a specific capture probe,e.g., pRNA partner specifically binding a pRNA (i.e., capture moiety)contained on a capture probe (i.e., antibody specific for a targetantigen).

In various embodiments, the plurality of specific binding agentscomprise a multitude of groups of specific binding pairs, wherein eachgroup comprises binding agents that specifically bind one targetantigen, and a second group of binding pairs which specifically bind asecond different antigen. Thus a plurality of specific binding pairscomprised in a SCD are capable of detecting a plurality of differenttarget analytes.

In various embodiments, the capture moiety is an oligonucleotide,av:idin, streptavidin, pyranosyl RNA (pRNA), aptamer, or a combinationthereof. In various embodiments, the label is a metal, a fluorophore, achromophore or a combination thereof. In some embodiments, the pluralityof specific binding pairs comprised in a SCD can contain one type ofcapture moiety but with different capture moiety partners, e.g., eachspecific binding agent conjugated to pRNA, where each group that isspecific to a different antigen comprises different pRNAs. In otherembodiments, the plurality of specific binding pairs comprises one ormore different capture moieties, e.g., pRNA for one group of specificbinding pairs, while streptavidin for another, or a combination ofdifferent types of capture moieties.

In some embodiments, the plurality of specific binding pairs comprisedin a SCD comprises one type of label (e.g., specific binding pairs whereeach group is conjugated to fluorophores having the same or fluorophoreshaving different wavelength signals). In other embodiments, specificbinding pairs comprise a combination of different types of labels (e.g.,combination of metals and fluorophores). In one embodiment, the capturemoiety is pRNA and the label is Europium.

In some embodiments, systems, devices and methods of the invention areconfigured to detect different analytes (i.e., target analytes; e.g.,FIG. 5A, 55 and 54) which are from an infectious agent. Examples ofinfectious agents include but are not limited to yeast, fungus,bacteria, virus, and parasitic organisms. It should be understood thatdepending on the target analyte sought to be detected, an antibody orother specific binding agent is utilized (e.g., antibody, aptamer) whichis specific for the particular target analyte. For example, antibodiescan be raised to whatever the target analyte and incorporated into thesystems, devices and methods of the invention.

In other embodiments, target analytes are infectious agents orcomponents thereof, including but not limited to virus or viralcomponents, bacteria or bacterial components, yeast or yeast components,fungus or fungal components, parasites or parasitic components, or anycombination thereof.

In some embodiments, target analytes are virus or components of virus.In further embodiments, the different target analytes are from influenzavirus and subtypes of influenza virus. For example, the influenza virusthat can be detected is influenza virus A and B as well as subtypes ofinfluenza virus. In one embodiment, an assay device is configured fordetection of influenza virus strains A and B and subtypes of the formulaHxNy, wherein x can be 1-16 and y can be 1-9, or any combination of xythereof.

In one embodiment, a method is provided for determining whether asubject is infected with a pandemic, non-pandemic or strain of influenzavirus for which vaccine is available.

In some embodiments, the Test Device excludes any reagent or bindingagent that is capable of specifically binding a target analyte. In otherwords, the TD does not comprise an entity that specifically binds atarget analyte.

In one aspect, a point of care (“POC”) assay is provided based onlateral flow of fluid through nitrocellulose while employing techniquesto reduce the background, improve the sensitivity and specificity of theassay. The detection technology employs a fluorescence detection systemusing a fluorophore. The fluorophore is coupled with a new capturesystem that enables the homogeneous binding of multiple monoclonalreagents to specific analytes and captures the complex onto specifictest lines. More detail in this regard is provided in related U.S.application Ser. No. 11/677,559.

In one aspect of invention, an assay is provided which is capable ofdetecting one or more analytes at a sensitivity comparable to tissueculture infectious does 50 (TCID₅₀), where sensitivity is measurable atdilutions from about 1:100, about 1:1000, about 1:10,000, about1:100,000 to about 1:1,000,000. In some embodiments, assays of theinvention provide sensitivity measured from TCID₅₀ at about 0.003,0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075,0.008, 0.0085, 0.009, 0.0095, 0.010, 0.0105, 0.020, 0.025, 0.030, 0.035,0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085,0.090, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.25,1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5,4.75, 5.0, 5.25, 5.50, 5.75, 6.0, 6.25, 6.50, 6.75, 7.0, 7.25, 7.5,7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75 or about 10.0. Infurther embodiments, TCID₅₀ is from about 25, 50, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, to about 600 per mL.

In one embodiment, Type A and Type B analytes show at least two logsimprovement in analyte sensitivity over a prior art POC Influenza assay.

In another aspect of the invention, the interface with the end user hasbeen redeveloped resulting in a fully integrated sample collectiondevice (SCD). These improvements enable high sensitivity multianalytedetection of a broad spectrum of analytes. In one embodiment, a singlediagnostic assay simultaneously and separately detects the majorsubtypes of Influenza Type A and Type B, but also seasonal subtypes(I-IINI and H3N2), and pandemic subtype H5N1. This will provide theclinician and public health agency important real time or near real timeinformation on influenza infection rates.

In one embodiment, for ease of use in a POC setting, a reader has beendesigned and has incorporated a simple to use icon driven interface.This will ensure that assays are recorded and frees personnel from thetask of assay interpretation. This third generation POC assay isintended to simplify POC testing while moving towards more reliableresults. Additional embodiments are disclosed in related U.S. patentapplication Ser. No. 11/677,559.

Sample Collection Device (SCD)

In one aspect of the invention a SCD is utilized to process a sample toallow detection of one or more analytes. For example, a SCD canincorporate a solid reagent component (e.g., capture and detectionprobes). Solid reagent components include, a powder, pill, bead,lyophilized pellet, pressed lyophilized power, dried on solid support(e.g., glass/plastic bead), lyophilized on or in association with asolid support or dried directly in the mixing or lower chamber. Suchreagents are known in the art such as disclosed in CURRENT PROTOCOLS INIMMUNOLOGY (Coligan, John E. et. al., eds. 1999).

In one embodiment of the invention, an SCD contains the following: A setof specimen extraction reagents in two separate reservoirs within the“handle” of the sample collection device. A lyophilized single usereagent or conjugate bead containing both capture and detectionreagents. The capture MAb(s) for each analyte are conjugated to sequencespecific pRNA capture molecules and detection MAb(s) which areconjugated to fluorescent microbeads.

In one embodiment an SCD comprises three components: (1) A set of swabsfor collecting a nasopharyngeal, nasal or throat specimen; (2) A handlewith extraction reagents containing with a valve mechanism (e.g., snapvalve or puncurable seal) to release the reagents at the appropriatestep in the assay; and (3) A plastic tube to contain the swab and thelyophilized reagent bead and a dropper tip (covered by a cap) to enabledirect connection of the SCD to the Test Device (TD).

In another embodiment, the distal end of the SCD is open, whereby priorto release of a solution from the upper sealed chamber, the SCD isengaged (e.g., by friction fit) into the receiving port of a TestDevice. In such an embodiment, the fluid flow from the distal end of theSCD into the Test Device is not regulated by a Iuer or a valvestructure.

In another embodiment, the distal end of the SCD does not utilize avalve but rather is open. The SCD may be attached to the test deviceprior to release of the buffer from the upper chamber. Upon release ofthe solution from the upper chamber, the sample is released and/orextracted from the collection implement by the solution and mixed withthe reagents located in the lower chamber. The mixture then flows to thetest device for analysis of the presence of one or more analytes. It ispossible to include water-dissolvable membranes within the lower chamberto slow the flow of the mixture out of the SCD onto the test device.Such membranes are conventional and can be designed to permit theretention of the mixture for differing periods of time sufficient toallow mixing and reaction of the reagents and sample analytes. Forexample, such membranes can be prepared from proteins, polysaccharidesor film formers.

In another embodiment, the distal end of an SCD comprises a very narrowopening that prevents fluid flow unless and until pressure is applied tothe device (e.g., via the bulb structure of the SCD, or if the housingis depressible, then by exerting pressure on the housing itself) toforce the fluid out from the distal end. In other words, there is novalve of any sort disposed at the distal end of the SCD

In some embodiments, the upper portion of the plastic tube has affixed alabel flag that incorporates two removable barcodes, one that must beplaced on the test device and one that may be place on the patientrecord. Additional embodiments are disclosed in related U.S. patentapplication Ser. No. 11/677,559.

In one embodiment, as shown in FIG. 1, the SCD contains the following: asample tube 10 with a cap 23, an ampoule 11 containing liquid withantiseptic agent 12, such as sodium azide, a sampling implement 13 and adropper cap 14. The conical tube may contain liquid 22 helpful forextraction of biological sample. In further embodiments, the dropper capfits on the sample tube. The dropper cap is configured with an outlet 17and a cap stopper 18 through which the content of the sample tube can bedispensed into sample receiving portion of Test Device 19. In furtherembodiments, lyophilized immunoreagents are added to the extractionmixture 22, or can be provided in a reagent chamber proximal to the openend of the SCD 10 or can be provided in the dropper cap 14. In oneembodiment, the dropper cap contains lyophilized reagent inside of thedropper cap 14 (e.g., capture and detection probes to one or moredifferent target analytes, 15, 16). In a further embodiment a screen(e.g., mesh or filter paper) is positioned 20 in the dropper cap. Forexample, the lyophilized reagents are held inside the dropper cap by ascreen mesh 20, and/or the screen can function as a size-based oraffinity-based filter of the extraction mixture 22.

In one embodiment, the SCD is configured with an compartment (e.g.,ampoule or releasable compartment) 11 containing reagents (e.g.,extraction buffer, and/or capture and detection probes). For example,during sample collection, the content of the compartment 11 can bedispensed into the sample tube 10. Furthermore, a sampling implement 21(e.g., swab, cotton tip, needle) is used to obtain a cell, tissue,bodily fluid or other sample. The sampling implement is subject to thecontents of the compartment 11 which contents can be released by theoperator of the device.

In one embodiment, the sample tube is capped with the dropper cap 14 andinverted to provide filtering via the membrane 20 and/or to allow mixingwith additional reagents 15, 16 provided in the dropper cap 14. A sampleis dispensed through outlet port 17 into a Test Device 19.

Reagents utilized in an SCD of the invention can include one or moresalts, chelators, anticoagulants, detergents, stabilizers, diluents,buffering agents, enzymes, cofactors, specific binding members, labels,mucolytic and the like. The one or more reagents can be compounds thatfacilitate analysis of a sample. Furthermore, such reagents can readilybe adapted for use in a Test Device of the invention. In variousembodiments, such reagents can be added to the sample tube 10 by theoperator or can be contained in compartment 11 which is configured toform a screw type or friction fit with the sample tube 10 before releaseof the compartment 11 contents. Alternatively, reagents 22 can bepresent in the sample tube 10 with a punctureable seal, so that a sampleimplement can be used to subject the contents 22 to contact with thesample present on the sampling implement 22.

In one embodiment, an extraction buffer includes, (1.5× buffer): 0.075MTris-Cl pH 8.5, 1.125M NaCl, 1.5% bovine serum albumin, 0.75% digestedcasein, 1.5% saponin, 0.375% zwittergent 3/12, 50 ug/ml gentamicinsulfate, 0.095% sodium azide, 0.025 mg/ml mouse IgG, 0.004% FD&C blue1,0.015% silicone antifoam. An extraction buffer provides enhancedextraction of a target analyte(s) (e.g., proteins or peptides from aninfectious agent). In further embodiments, a composition of extractionbuffer is comprised of a pH balancing agent, such as Tris-Cl, and otheragents helpful for complete extraction of target moiety from theanalytes, such as salt, serum, detergent, antibiotics, protein substratesuch as casein and antiseptic agent. In yet other embodiments, anextraction buffer comprises a salt in a range from 0.5M to 1.5M,including but not limited to 0.5M NaCl, 0.6M NaCl, 0.7M NaCl, 0.8M NaCl,0.9M NaCl, 1.0 M NaCl, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M; or from 0.8M to1.5M, including but not limited to 0.85M NaCl, 0.95M NaCl, 1.05M NaCl,1.1M NaCl, 1.2M NaCl, 1.3M NaCl, 1.4M NaCl, and 1.5M NaCl.

In other embodiments, extraction buffer further comprise saponin, in anamount ranging from 0.25% to 1.5%, including but not limited to about0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.3%,1.4%, 1.5%, or from 0.03% to 0.08%, such as 0.0375%, 0.045%, 0.0475%,0.05%, 0.055%, 0.0575%, 0.06%, 0.065%, 0.0675%, 0.07%, 0.075%, 0.08%. Inone embodiment, saponin is from about 1% to 2%, including but notlimited to about 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% toabout 2.0%.

Test Device (TD)

As used herein in the context of the Test Device (e.g., FIG. 1, 19; FIG.2) the terms “axial flow membrane”, “lateral flow membrane”, “testmembrane”, “test strip” or “matrix” are used interchangeably whichemploys capillary action to move or transport the test fluids or employsthe movement of fluid separate from capillary action as where fluid ispumped by the accumulation of gas pressure, hydraulic pressure (directpumping using a piston or rotary, bellows or other type pump on theassay fluids, electrostatic movement due to an electric field, gravity,etc.).

Another aspect of the invention is directed to a TD (FIG. 2) comprisinga plastic housing with a port to which the SCD is easily affixed, awindow for the reader to scan the rest result, and a button that theoperator depresses after removing the SCD. In one embodiment a TDcomprises: (I) A lateral flow test strip with a wicking membrane toadsorb the extracted specimen/reagent mixture; (2) A nitrocellulosestrip with multiple lines. Each test line on the nitrocelllulose teststrip is striped with a unique pRNA (e.g., FIG. 5A: 56, 57, 58, 59),which captures its homologous binding pair with the specific captureagent (e.g., antibody) 60 attached; (3) An adsorbent pad 50 providessupport the flow of fluid across the membrane; alternatively, the TestDevice FIG. 2 comprises a (4) A buffer packet that is ruptured upondepressing the button allowing wash buffer to follow the specimen acrossthe nitrocellulose pad aiding in the reduction of background andstopping the assay. Additional embodiments are disclosed in related U.S.patent application Ser. No. 11/677,559.

In one aspect, the signal obtained by the reader is processed using dataprocessing software employing data reduction and curve fittingalgorithms, optionally in combination with a trained neural network, togive either a positive or negative result for each test line, or aquantitative determination of the concentration of each analyte in thesample, which is correlated with a result indicative of a risk orpresence of a disease or disorder. This result can optionally be inputinto a decision support system, and processed to provide an enhancedassessment of the risk of a medical condition as output. In oneembodiment, the entire procedure may be automated and/or computercontrolled.

In one embodiment, the liquid transport along the test strip is basedupon capillary action. In a further embodiment, the liquid transportalong the matrix is based on non-bibulous lateral flow, wherein all ofthe dissolved or dispersed components of the liquid sample are carriedat substantially equal rates and with relatively unimpaired flowlaterally through the matrix, as opposed to preferential retention ofone or more components as would occur, e.g., in materials that interact,chemically, physically, ionic ally or otherwise with one or morecomponents. See for example, U.S. Pat. No. 4,943,522, herebyincorporated by reference in its entirety.

Any suitable material can be used to make the devices disclosed herein,such material including a rigid or semi-rigid, non-water-permeablematerial, such as glass, ceramics, metals, plastics, polymers, orcopolymers, or any combination thereof. In some embodiments, either theSCD or Test Device comprise a plastic, polymer or copolymer such asthose that are resistant to breakage, such as polypropylene,polyallomer, polycarbonate or cycloolefins or cycloolefin copolymers.Furthermore, devices of the invention can be made by appropriatemanufacturing methods, such as, but not limited to, injection molding,blow molding, machining or press molding.

As used herein, test strip substrate refers to the material to which acapture moiety is linked using conventional methods in the art. Avariety of materials can be used as the substrate, including anymaterial that can act as a support for attachment of the molecules ofinterest. Such materials are known to those of skill in this art andinclude, but are not limited to, organic or inorganic polymers, naturaland synthetic polymers, including, but not limited to, agarose,cellulose, nitrocellulose, cellulose acetate, other cellulosederivatives, dextran, dextran derivatives and dextran co-polymers, otherpolysaccharides, glass, silica gels, gelatin, polyvinyl pyrrolidone(PVP), rayon, nylon, polyethylene, polypropylene, polybutlyene,polycarbonate, polyesters, polyarnides, vinyl polymers,polyvinylalcohols, polystyrene and polystyrene copolymers, polystyrenecross-linked with divinylbenzene or the like, acrylic resins, acrylatesand acrylic acids, acrylamides, polyacrylamide, polyacrylamide blends,co-polymers of vinyl and acrylamide, methacrylates, methacrylatederivatives and co-polymers, other polymers and co-polymers with variousfunctional groups, latex, butyl rubber and other synthetic rubbers,silicon, glass, paper, natural sponges, insoluble protein, surfactants,red blood cells, metals, metalloids, magnetic materials, or othercommercially available media or a complex material composed of a solidor semi-solid substrate coated with materials that improve thehydrophilic property of the strip substrate, for example, polystyrene,Mylar, polyethylene, polycarbonate, polypropylene, polybutlyene, metalssuch as aluminum, copper, tin or mixtures of metals coated with dextran,detergents, salts, PVP and/or treated with electrostatic or plasmadischarge to add charge to the surface thus imparting a hydrophilicproperty to the surface.

In one embodiment, the lateral flow membrane is comprised of a porousmaterial such as high density polyethylene sheet material manufacturedby Porex Technologies Corp. of Fairburn, Ga., USA. The sheet materialhas an open pore structure with a typical density, at 40% void volume,of 0.57 gm/cc and an average pore diameter of 1 to 250 micrometers, theaverage generally being from 3 to 100 micrometers. In anotherembodiment, the label zone is comprised of a porous material such as anonwoven spun laced acrylic fiber (similar to the sample receivingzone), e.g., New Merge or HDK material. Often, the porous material maybe backed by, or laminated upon, a generally water impervious layer,e.g., Mylar. When employed, the backing is generally fastened to thematrix by an adhesive (e.g., 3M 444 double-sided adhesive tape).Typically, a water impervious backing is used for membranes of lowthickness. A wide variety of polymers may be used provided that they donot bind nonspecifically to the assay components and do not interferewith flow of the fluid sample. Illustrative polymers includepolyethylene, polypropylene, polystyrene and the like. On occasion, thematrix may be self-supporting. Other membranes amenable to non-bibulousflow, such as polyvinyl chloride, polyvinyl acetate, copolymers of vinylacetate and vinyl chloride, polyamide, polycarbonate, polystyrene, andthe like, can also be used. In yet another embodiment, the lateral flowmembrane is comprised of a material such as untreated paper, celluloseblends, nitrocellulose, polyester, an acrylonitrile copolymer, and thelike. The label zone may be constructed to provide either bibulous ornonbibulous flow, frequently the flow type is similar or identical tothat provided in at least a portion of the sample receiving zone. In afrequent embodiment, the label zone is comprised of a nonwoven fabricsuch as Rayon or glass fiber. Other label zone materials suitable foruse by the present invention include those chromatographic materialsdisclosed in U.S. Pat. No. 5,075,078, which is herein incorporated byreference.

In a frequent embodiment, the test strip substrate is treated with asolution that includes material-blocking and label-stabilizing agents.Blocking agents include bovine serum albumin (BSA), methylated BSA,casein, acid or base hydrolyzed casein, nonfat dry milk, fish gelatin,or similar. Stabilizing agents are readily available and well known inthe art, and may be used, for example, to stabilize labeled reagents. Insome embodiments, the upstream compartment containing a solution cancomprise multiple ampoules, which can be selectively punctured or brokento release their contents. Therefore, in one embodiment, blockingreagents are contained in one ampoule which is utilized to pre-treat(e.g., “block”) the test strip (i.e., lateral flow membrane), while theadditional ampoule is reserved for washing the sample through the teststrip.

The signal obtained by the reader is processed using data processingsoftware employing data reduction and curve fitting algorithms,optionally in combination with a trained neural network, to give eithera positive or negative result for each test line, or a quantitativedetermination of the concentration of each analyte in the sample, whichis correlated with a result indicative of a risk or presence of adisease or disorder. This result can optionally be input into a decisionsupport system, and processed to provide an enhanced assessment of therisk of a medical condition as output. In one embodiment, the entireprocedure may be automated and/or computer-controlled.

As used herein, test strip substrate refers to the material to which acapture moiety is linked using conventional methods in the art. Avariety of materials can be used as the substrate, including anymaterial that can act as a support for attachment of the molecules ofinterest. Such materials are known to those of skill in this art andinclude, but are not limited to, organic or inorganic polymers, naturaland synthetic polymers, including, but not limited to, agarose,cellulose, nitrocellulose, cellulose acetate, other cellulosederivatives, dextran, dextranderivatives and dextran co-polymers, otherpolysaccharides, glass, silica gels, gelatin, polyvinyl pyrrolidone(PVP), rayon, nylon, polyethylene, polypropylene, polybutlyene,polycarbonate, polyesters, polyamides, vinyl polymers,polyvinylalcohols, polystyrene and polystyrene copolymers, polystyrenecross-linked with divinylbenzene or the like, acrylic resins, acrylatesand acrylic acids, acrylamides, polyacrylamide, polyacrylamide blends,co-polymers of vinyl and acrylamide, methacrylates, methacrylatederivatives and co-polymers, other polymers and co-polymers with variousfunctional groups, latex, butyl rubber and other synthetic rubbers,silicon, glass, paper, natural sponges, insoluble protein, surfactants,red blood cells, metals, metalloids, magnetic materials, or othercommercially available media or a complex material composed of a solidor semi-solid substrate coated with materials that improve thehydrophilic property of the strip substrate, for example, polystyrene,Mylar, polyethylene, polycarbonate, polypropylene, polybutlyene, metalssuch as aluminum, copper, tin or mixtures of metals coated with dextran,detergents, salts, PVP and/or treated with electrostatic or plasmadischarge to add charge to the surface thus imparting a hydrophilicproperty to the surface.

In one embodiment, a test strip substrate is treated with a solutionthat includes material-blocking and label stabilizing agents. Blockingagents include bovine serum albumin (BSA), methylated BSA, casein, acidor base hydrolyzed casein, nonfat dry milk, fish gelatin, or similar.Stabilizing agents are readily available and well known in the art, andmay be used, for example, to stabilize labeled reagents. In someembodiments, the upstream compartment containing a solution can comprisemultiple ampules, which can be selectively punctured or broken torelease their contents. Therefore, in one embodiment, blocking reagentsare contained in one ampule which is utilized to pre-treat (e.g.,“block”) the test strip (i.e., lateral flow membrane), while theadditional ampule is reserved for washing the sample through the teststrip.

The signal obtained by the reader is processed using data processingsoftware employing data reduction and curve fitting algorithms,optionally in combination with a trained neural network, to give eithera positive or negative result for each test line, or a quantitativedetermination of the concentration of each analyte in the sample, whichis correlated with a result indicative of a risk or presence of adisease or disorder. This result can optionally be input into a decisionsupport system, and processed to provide an enhanced assessment of therisk of a medical condition as output. In one embodiment, the entireprocedure may be automated and/or computer-controlled.

In a frequent embodiment, the test strip substrate is treated with asolution that includes material-blocking and label-stabilizing agents.Blocking agents include bovine serum albumin (BSA), methylated BSA,casein, acid or base hydrolyzed casein, nonfat dry milk, fish gelatin,or similar. Stabilizing agents are readily available and well known inthe art, and may be used, for example, to stabilize labeled reagents. Insome embodiments, the upstream compartment containing a solution cancomprise multiple ampules, which can be selectively punctured or brokento release their contents. Therefore, in one embodiment, blockingreagents are contained in one ampule which is utilized to pre-treat(e.g., “block”) the test strip (i.e., lateral flow membrane), while theadditional ampule is reserved for washing the sample through the teststrip.

Exemplary functions of the labeled control reagents and zones include,for example, the confirmation that the liquid flow of the sampleeffectively solubilized and mobilized the labeled reagents from the SCD,which are captured in one or more defined test zones. Furthermore,controls can confirm that a sufficient amount of liquid traveledcorrectly through the test strip test and control zones, such that asufficient amount of capture moieties could react with the correspondingspecific capture probes complexed to a specific analyte (i.e., via theantigen specific binding agent). Further, control reagents confirm thatthe immunocomplexes (e.g., analyte-analyte specific binding agent)migrate onto the test region comprising the test and control zones,cross the test zone(s) in an amount such that the accumulation of thelabeled analyte would produce a visible or otherwise readable signal inthe case of a positive test result in the test zone(s). Moreover, anadditional function of the control zones may be to act as referencezones which allow the user to identify the test results which aredisplayed as readable zones.

Since the devices of the present invention may incorporate one or morecontrol zones, the labeled control reagent and their correspondingcontrol zones are preferably developed such that each control zone willbecome visible with a desired intensity for all control zones afterfluid sample is contacted with the device, regardless of the presence orabsence of one or more analytes of interest.

In one embodiment, a single labeled control reagent will be captured byeach of the control zones on the test strip. Frequently, such a labeledcontrol reagent will be deposited onto or in the label zone in an amountexceeding the capacity of the total binding capacity of the combinedcontrol zones if multiple control zones are present. Accordingly, theamount of capture reagent specific for the control label can bedeposited in an amount that allows for the generation of desired signalintensity in the one or more control zones, and allows each of thecontrol zones to restrain a desired amount of labeled control-reagent.At the completion of an assay, each of the control zones preferablyprovide a desired and/or pre-designed signal (in intensity and form).Examples of contemplated pre-designed signals include signals of equalintensities in each control zone, or following a desired pattern ofincreasing, decreasing or other signal intensity in the control zones.

In another embodiment, each control zone will be specific for a uniquecontrol reagent. In this embodiment, the label zone may include multipleand different labeled control reagents, equaling the number of controlzones in the assay, or a related variation. Wherein each of the labeledcontrol reagents may become restrained in one or more pre-determined andspecific control zone(s). These labeled control reagents can provide thesame detectable signal (e.g., be of the same color) or providedistinguishable detectable signals (e.g., have different colored labelsor other detection systems) upon accumulation in the control zone(s).

In yet another embodiment, the control zones may include a combinationof the two types of control zones described in the two previousembodiments, specifically, one or more control zones are able torestrain or bind a single type of labeled control reagent, and othercontrol zones on the same test strip will be capable of binding one orseveral other specifically labeled control reagents.

In one embodiment, the labeled control reagent comprises a detectablemoiety coupled to a member of a specific binding pair. Typically, alabeled control reagent is chosen to be different from the reagent thatis recognized by the means which are capable of restraining an analyteof interest in the test zone. Further, the labeled control reagent isgenerally not specific for the analyte. In a frequent embodiment, thelabeled control reagent is capable of binding the corresponding memberof a specific binding pair or control capture partner that isimmobilized on or in the control zone. Thus the labeled control reagentis directly restrained in the control zone.

In another embodiment, the detectable moiety which forms the labelcomponent of the labeled control reagent is the same detectable moietyas that which is utilized as the label component of the analyte ofinterest labeled test reagent. In a frequent embodiment, the labelcomponent of the labeled control reagent is different from the labelcomponent of the labeled test reagent, so that results of the assay areeasily determined. In another frequent embodiment, the control label andthe test label include colored beads, e.g., colored latex. Alsofrequently, the control and test latex beads comprise different colors.

In a further embodiment, the labeled control reagent includesstreptavidin, avidin or biotin and the control capture partner includesthe corresponding member of such specific binding pairs, which readilyand specifically bind with one another. In one example, the labeledcontrol reagent includes biotin, and the control capture partnerincludes streptavidin. The artisan will appreciate that other members ofspecific binding pairs can alternatively be used, including, forexample, antigen/antibody reactions unrelated to analyte. In yet otherembodiment, capture partners can include any of the binding moietiesdisclosed herein.

The use of a control zone is helpful in that appearance of a signal inthe control zone indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in a test zonecan be noted.

In still further embodiment, a control zone comprising a mark thatbecomes visible in the test region when the test region is in a moiststate is utilized. Control zones of this type are described in U.S.patent application Ser. No. 09/950,366, filed, Sep. 10, 2001, currentlypending and published as U.S. patent application Publication No.20030049167, and Ser. No. 10/241,822, filed Sep. 10, 2002, currentlypending and published as U.S. patent application Publication No.20030157699.

In some embodiments, one or more control zones of this type areutilized. In another embodiment, a combination of control zones of thetype utilizing labeled control reagents and control zone and of the typethat display the control zone when in a moist state can be used. Thisallows a simple way to formulate control zones while allowing to use areagent-based control zone to ascertain that the re-solubilization andmobilization of the reagents in SCD-processed samples has beeneffective, and that the specific reactions took place as expected, allalong the path defined Test Device, wick, test strip and absorbent pad.The present embodiment includes the use of one or more control zonesthat become visible when the test region is in the moist state for eachof the control zones of an assay, except the control zone on the distalor downstream end of the test strip.

The present description further provides means to build a rapid,multi-analyte assay, which is needed in many fields of environmentalmonitoring, medicine, particularly in the field of infectious disease.For example, contemplated devices include those useful for thedifferential diagnosis of Flu A or Flu B, and subtypes thereof (e.g.,Flu A, H5N1) which may result in different treatments, or thedifferential diagnosis of Flu A, Flu B, and/or RSV in one step. Suchdevices permit the use of a single sample for assaying multiple analytesat once, and beneficially allows for a considerable reduction of thehands-on time and duration of the diagnostic process for the benefit ofthe doctor, or user in general. As such a plurality of immunoreagentscan be utilized in an SCD of the invention, where said pluralitycomprises populations of specific binding agents, comprising pairsconjugated respectively to label and capture moiety, whereby saidplurality comprise multiple populations each specific for a differentanalyte as compared to any other population. For example, the pluralityof immunoreagents can be specific for several types of one pathogen ordifferent pathogens.

A variety of analytes may be assayed utilizing devices and methods ofthe present disclosure. In a particular device useful for assaying forone or more analytes of interest in a sample, the collection of analytesof interest may be referred to as a panel. For example, a panel maycomprise any combination (or all of) of influenza A, influenza B,influenza A subtypes, respiratory syncytial virus (RSV), adenovirus, anddifferent types of Parainfluenza viruses (for example Types 1,2,3 etc.).Another panel may comprise testing for a selection of one or more ofupper respiratory infection including, for example, Streptococcuspneumoniae, Mycoplasma pneumoniae and/or Chlamydia pneumoniae. Yetanother panel can be devised for the diagnosis of sexually transmitteddisease including, for example, Chlamydia, Trichomonas and/or Gonorrhea.In each case, a particular panel devised to provide signals on the TestDevice for a particular series of analytes is readily obtained byincorporating a different set of detection and capture probes in theSCD, which is described herein. Therefore, a particular SCD will provideall the reagents necessary to detect a particular panel of analyteswhich are detected when using a Test Device employing test strips thathave detection reagents that are not specific for the analytes ofinterest. In other embodiments, a broad scope Test Device can comprisenon-specific capture moieties for several series of analytes fromrelated or distinct pathogens, e.g., detection of HIV and HCV antigens;HIV and tuberculosis, Influenza A, B, and subtypes of A, bacterial andviral infections. Thus a single Test Device can be used with SCDscomprising immunoreagents for a different panel of analytes, providingenhanced efficiency and cost effectiveness.

For example, a panel may optionally include a variety of other analytesof interest, including SARS associated coronavirus, influenza A; ahepatitis panel comprising a selection of hepatitis B surface Ag or Ab,hepatitis B core Ab, hepatitis A virus Ab, and hepatitis C virus; aphospholipids panel comprising a selection of Anticardiolipin Abs (IgG,IgA, and IgM Isotypes); an arthritis panel comprising a selection ofrheumatoid factor, antinuclear antibodies, and Uric Acid; an EpsteinBarr panel comprising a selection of Epstein Barr Nuclear Ag, EpsteinBarr Viral Capsid Ag, and Epstein Barr Virus, Early Antigen; otherpanels include HIV panels, Lupus panels, H. Pylori panels, toxoplasmapanels, herpes panels, Borrelia panels, rubella panels, cytomegaloviruspanels, panels testing for recent myocardial infarction with analytescomprising an isotype of Troponin with myoglobin and/or CKMB and manyothers. One of skill in the art would understand that a variety ofpanels may be assayed via the immunoassays utilizing the devicesdisclosed herein. Immunoassay methods are known in the art. See, e.g.,CURRENT PROTOCOLS IN IMMUNOLOGY (Coligan, John E. et. al., eds. 1999).

Numerous analytical devices known to those of skill in the art may beadapted in accordance with the present invention, to detect multipleanalytes. By way of example, dipstick, lateral flow and flow-throughdevices, particularly those that are immunoassays, may be modified inaccordance herewith in order to detect and distinguish multipleanalytes. Exemplary lateral flow devices include those described in U.S.Pat. Nos. 4,818,677, 4,943,522, 5,096,837 (RE 35,306), 5,096,837,5,118,428, 5,118,630, 5,221,616, 5,223,220, 5,225,328, 5,415,994,5,434,057, 5,521,102, 5,536,646, 5,541,069, 5,686,315, 5,763,262,5,766,961, 5,770,460, 5,773,234, 5,786,220, 5,804,452, 5,814,455,5,939,331, 6,306,642. Other lateral flow devices that may be modifiedfor use in distinguishable detection of multiple analytes in a fluidsample include U.S. Pat. Nos. 4,703,017, 6,187,598, 6,352,862,6,485,982, 6,534,320 and 6,767,714. Exemplary dipstick devices includethose described in U.S. Pat. Nos. 4,235,601, 5,559,041, 5,712,172 and6,790,611. It will be appreciated by those of skill in the art that theaforementioned patents may and frequently do disclose more than oneassay configuration and are likewise referred to herein for suchadditional disclosures. Advantageously, the improvements described areapplicable to various assay, especially immunoassay, configurations.

Reader

In yet a further aspect of the invention a reader provides effectivedetection. In one embodiment, an icon driven fluorescent reader (FIG. 3)is required to read the bar coded information on the TD, ensuring thecomponents are in date and returns a result for each analyte line.Additional embodiments are disclosed in related U.S. patent applicationSer. No. 11/677,559.

Multianalyte

In one aspect of the invention, one or more analytes are detected usingmethods, systems and devices of the invention. As disclosed herein, asample can be from any source and the invention is configured to provideenhanced sensitivity and specificity for detecting an analyte, whileproviding capabilities to detect a plurality of different analytes. Thetype of analytes that can be detected are discussed herein.

Rapid influenza tests have been marketed for years. Most of these testsare lateral flow immunoassay tests using either gold or latex as thevisualization agent. While most of new rapid immunoassays are able todifferentiate influenza Type A from influenza Type B, only few of themhave both test lines for type A and type B on the one strip. However,none of these tests are designed to differentiate subtypes of influenzatype A. Therefore these tests may be able to detect avian influenza,none of them can tell if a patient is infected by a seasonal flu A virusor a more severe Type A subtype such as H5N1 termed avian influenza (orcurrent potential pandemic subtype of influenza A). The presentinvention is designed on concepts that when applied are to yield ahighly sensitive assay with improved reproducibility, able to detecttype A, type B and differentiate subtype H5N1 from seasonal flu(subtypes H1 and H3) and is easy to use. Efforts have been made to applymultiple new technologies with a new device design, such as pre-mixingof the sample with the conjugate, use of a chasing or wash buffer toreduce background, employ a unique generic capture reagent pRNA whichallows multiple analytes detection at high sensitivity, fluorescentlabel which is highly sensitive, etc. The combination of theseapproaches enables a novel and highly effective influenza rapid testthat is much more sensitive, provides low cost production, ease ofoperate and has the ability to differentiate seasonal flu from pandemicavian flu H5N1. In one embodiment, the combination of features describedherein are responsible for the excellent sensitivity and reproducibilityof assays constructed in accordance with the invention to use the novelsystem, which serves to concentrate ligand from a test sample at a testsite the test strip, and the use of a metal sol or other coloredparticle as a marker system which permits persistent visual observationof the fluorescence over a period of one to several hours beyond theminimum time needed to complete the assay). Background noise is reducedwhile maintaining excellent sensitivity by including in the testimplement a controllably released buffer that functions to wash awayexcess/unbound label. Furthermore, one or more control sites whose coloris compared with the test site. In some embodiments, a filtration meansis comprised in the sample implement or in the test implement, whichfiltration means limits the introduction to the test site ofcontaminants from the crude or unprocessed biological sample.

Assay Methods.

In one embodiment, an assay method comprises the steps of applying thesampling implement to a subject or subject's biological sample, tocollect a sample (e.g., swabbing inside the nose, mouth, throat, ear;applying a sampling element to a biological sample obtained from asubject), inserting the collection implement into the sample collectiondevice housing chamber, squeezing the upper chamber to break open thesnap-valve and allowing a buffer to run down to the sampling implement,thus immersing the biological sample disposed thereon and running themixture of buffer and sample into a reaction chamber (e.g., lowerchamber) where a plurality of capture and detection probes bind to theirspecific target analyte. Subsequently or concurrently, the mixture isexpelled from the distal end of the SCD into a Test Device comprisingimmobilized capture moieties designed to capture a complex of analyteand detection/capture probe via the complementary capture moiety linkedto a capture probe. Thus, a particular capture probe is designed to becomplementary to an immobilized capture moiety for one particularanalyte. Furthermore, as disclosed herein, capture moieties are disposedon a lateral flow membrane in distinct positions/patterns, where asingle line or spot(s) if detected via the signal emitting label, allowsqualitative and/or quantitative detection of a particular analyte.Therefore, by patterning particular capture probes on the lateral flowmembrane, an assay method can detect a panel of the same or relatedinfectious agent or even unrelated infectious agents, as disclosedherein.

In some embodiments a sandwich immunoassay format is utilized but anyconventional format, including a competitive assay, may be used.Typically, an indirect capture of the formed immunocomplex is utilizedin the sandwich format. One or more analytes in the sample are contactedwith one or more pairs of a detection probe and a capture probe. Eachpair contains the detection probe which is a conjugate comprising alabel and a specific binding agent (SBA) capable of specifically bindingto an analyte and a capture probe which is a conjugate comprising adetection moiety and another SBA capable of specifically binding thesame analyte. Examples of specific binding agent(s) include antibodies,aptamers, In one embodiment, each of two SBAs are specific bindingpartners in that each bind the same target antigen or analyte.

Examples of SBAs are known in the art and include but are not limited toantibodies, aptamers or oligonucleotides. In the sandwich assay, theanalyte is simultaneously bound by both the detection probe and thecapture probe. The detection moiety is part of a specific binding pairand the other partner of the pair is immobilized on the test device tocapture the immunocomplex as it flows through the test device. The useof different capture moiety pairs for each different analyte permits thedetection of multiple analyte on one test device from a single sampleand reaction sequence. In most cases, the labels for each analyte aredifferent. However, by having a specific location for each analyte as acapture zone with distinct capture moiety pairs for each analyte, it ispossible to utilize the same label for all of the analyte.

Binding Reagents

One aspect of the invention is directed to binding reagents disposed inthe SCD. For example, in some immunoassays, an antibody pair isutilized, where each member of the pair can specifically bind the sametarget analyte, wherein one antibody is a capture antibody and the otheris a detection antibody. A capture antibody is linked, directly orindirectly, to a capture moiety which is “captured” by a cognateimmobilized capture moiety disposed on a solid support (e.g.,nitrocellulose membrane). Furthermore, the detection antibody (i.e.,detection probe) is linked to a detectable label. The detection antibodyis preferably labeled by conjugation to a physically detectable label,and upon contacting with the sample containing the target analyte formsa complex. The antibody analyte complex can then be immobilized on asolid support via the capture moiety. The resulting complex immobilizedon the solid support, is detectable by virtue of the label.

In one embodiment, the SCD reagent solution or solid substrate comprisesa plurality of different detection probes, each detection probe capableof binding to a different target and each detection probe being labeledwith or enabling the formation of a detection signal so that thepresence of each target is indicated by the formation of a signal at thetest zone for that target (i.e., in the Test Device); wherein the targetfor at least two of the capture moieties is an infectious agent or adisease causing micro-organism or a marker indicating the existence of adisease, disorder, or condition of the host from which the samplesolution was derived, and wherein at least two of the capture moietiesare capable of binding to different components or markers of the sameinfectious agent or disease causing microorganism, or to differentmarkers for the same disease, disorder, or condition not caused by aninfectious agent or disease causing microorganism, as targets for thosecapture moieties. Furthermore, the SCD will also comprise a plurality ofdifferent capture probes, each of which is paired up with a detectionprobe, where the pairing is defined by the capability to bind aparticular target analyte.

As used herein, the term “specifically binds” refers to the bindingspecificity of a specific binding pair. “Specific binding pair member”refers to a member of a specific binding pair (“sbp”), which means twodifferent molecules wherein one of the molecules specifically binds withthe second molecule through chemical or physical means. For example, apair of pRNAs or an aptamer/target antigen pair, or streptavidin-biotinprovide exemplary specific binding pair members or sbp. The twomolecules are related in the sense that their binding with each other issuch that they are capable of distinguishing their binding partner fromother assay constituents having similar characteristics. The members ofthe specific binding pair are referred to as ligand and receptor(antiligand), sbp member and sbp partner, and the like. A molecule mayalso be a sbp member for an aggregation of molecules; for example anantibody raised against an immune complex of a second antibody and itscorresponding antigen may be considered to be a sbp member for theimmune complex.

In addition to antigen and antibody specific binding pair members, otherspecific binding pairs include, as examples without limitation, biotinand avidin, carbohydrates and lectins, complementary nucleotidesequences, complementary peptide sequences, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,a peptide sequence or chemical moiety (such as digoxin/anti-digoxin) andan antibody specific for the sequence, chemical moiety or the entireprotein, polymeric acids and bases, dyes and protein binders, peptidesand specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), metals and their chelators, and the like.Furthermore, specific binding pairs can include members that are analogsof the original specific binding member, for example an analyte-analogor a specific binding member made by recombinant techniques or molecularengineering.

A sbp member is analogous to another sbp member if they are both capableof binding to another identical complementary sbp member. Such a sbpmember may, for example, be either a ligand or a receptor that has beenmodified by the replacement of at least one hydrogen atom by a group toprovide, for example, a labeled ligand or labeled receptor. The sbpmembers can be analogous to or complementary to the analyte or to an sbpmember that is complementary to the analyte. If the specific bindingmember is an immunoreactant it can be, for example, an antibody,antigen, hapten, or complex thereof. If an antibody is used, it can be amonoclonal or polyclonal antibody, a recombinant protein or antibody, achimeric antibody, a mixture(s) or fragment(s) thereof, as well as amixture of an antibody and other specific binding members. Otherexamples of binding pairs that can be incorporated into the detectionmolecules are disclosed in U.S. Pat. Nos. 6,946,546, 6,967,250,6,984,491, 7,022,492, 7,026,120, 7,022,529, 7,026,135, 7,033,781,7,052,854, 7,052,916 and 7,056,679.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, andincludes any immunoglobulin, including monoclonal antibodies, polyclonalantibodies, multispecific or bispecific antibodies, that bind to aspecific antigen. A complete antibody comprises two heavy chains and twolight chains. Each heavy chain consists of a variable region and afirst, second, and third constant region, while each light chainconsists of a variable region and a constant region. The antibody has a“Y” shape, with the stem of the Y consisting of the second and thirdconstant regions of two heavy chains bound together via disulfidebonding. Each arm of the Y consists of the variable region and firstconstant region of a single heavy chain bound to the variable andconstant regions of a single light chain. The variable regions of thelight and heavy chains are responsible for antigen binding. The variableregion in both chains generally contains three highly variable loopscalled the complementarity determining regions (CDRs) (light (L) chainCDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs includingHCDR1, HCDR2, HCDR3) (as defined by Kabat, et al., Sequences of Proteinsof immunological Interest, Fifth Edition (1991), vols. 1-3, NIHPublication 91-3242, Bethesda Md.). The three CDRs are interposedbetween flanking stretches known as framework regions (FRs), which aremore highly conserved than the CDRs and form a scaffold to support thehypervariable loops. The constant regions of the heavy and light chainsare not involved in antigen binding, but exhibit various effectorfunctions. The recognized immunoglobulin genes include the kappa,lambda, alpha, gamma, delta, epsilon, and mu constant regions, as wellas myriad immunoglobulin variable region genes. Light chains areclassified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes and subclasses include IgG, IgG1, IgG2, IgG3,IgG4, IgM, IgA, IgA1, or IgA2, IgD, and IgE, respectively. Typically, anantibody is an immunoglobulin having an area on its surface or in acavity that specifically binds to and is thereby defied as complementarywith a particular spatial and polar organization of another molecule.The antibody can be polyclonal or monoclonal. Antibodies may include acomplete immunoglobulin or fragments thereof. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′, and the like. Antibodies may alsoinclude chimeric antibodies or fragment thereof made by recombinantmethods. The term “antibody” as used herein Antibodies are assigned toclasses based on the amino acid sequence of the constant region of theirheavy chain. The major classes of antibodies are IgA, IgD, IgE, IgG, andIgM, with several of these classes divided into subclasses such as.

In addition to an intact immunoglobulin, the term “antibody” as usedherein further refers to an immunoglobulin fragment thereof (i.e., atleast one immunologically active portion of an immunoglobulin molecule),such as a Fab, Fab′, F(ab′)z, Fv fragment, a single-chain antibodymolecule, a multispecific antibody formed from any fragment of animmunoglobulin molecule comprising one or more CDRs. In addition, anantibody as used herein may comprise one or more CDRs from a particularhuman immunoglobulin grafted to a framework region from one or moredifferent human immunoglobulins.

“Fab” with regards to an antibody refers to that portion of the antibodyconsisting of a single light chain (both variable and constant regions)bound to the variable region and first constant region of a single heavychain by a disulfide bond.

“Fab” refers to a Fab fragment that includes a portion of the hingeregion.

“F(ab′)2 refers to a dimer of Fab′.

“Fc” with regards to an antibody refers to that portion of the antibodyconsisting of the second and third constant regions of a first heavychain bound to the second and third constant regions of a second heavychain via disulfide bonding. The Fe portion of the antibody isresponsible for various effector functions but does not function inantigen binding.

“Fv” with regards to an antibody refers to the smallest fragment of theantibody to bear the complete antigen binding site. An Fv fragmentconsists of the variable region of a single light chain bound to thevariable region of a single heavy chain.

“Single-chain Fv antibody” or “scFv” refers to an engineered antibodyconsisting of a light chain variable region and a heavy chain variableregion connected to one another directly or via a peptide linkersequence (Houston 1988).

“Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineeredantibody consisting of a scFv connected to the Fe region of an antibody.

The term “epitope” as used herein refers to the group of atoms and/oramino acids on an antigen molecule to which an antibody binds.

The term “monoclonal antibody” as used herein refers to an antibody or afragment thereof obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single epitope on the antigen. Monoclonalantibodies are in contrast to polyclonal antibodies which typicallyinclude different antibodies directed against different epitopes on theantigens. Although monoclonal antibodies are traditionally derived fromhybridomas, the monoclonal antibodies of the present invention are notlimited by their production method. For example, the monoclonalantibodies of the present invention may be made by the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

The term “chimeric antibody” as used herein refers to an antibody inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass, as well as fragments of such an antibody, so long as suchfragments exhibit the desired antigen-binding activity (U.S. Pat. No.4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci.USA, 81:6851 6855 (1984>>.

The term “humanized antibody” used herein refers to an antibody orfragments thereof which are human immunoglobulins (recipient antibody)in which residues from part or all of a CDR of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity,and capacity. In some instances, FR residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues which are found neither in therecipient antibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin Fe region, typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321:522 525 (1986); Reichmannet al., Nature, 332:323 329 (1988); Presta, Curro Op. Struct. Biot,2:593 596 (1992); and Clark, Immunol. Today 21: 397402 (2000).

In various embodiments, a device of the invention is configured for usewith anti-H5 monoclonal antibodies that are produced by mice hybridomacell strains as disclosed in U.S. application Ser. No. 11/677,559.Antibodies are designed to specifically bind to the hemagglutinin ofsubtype H5 avian influenza virus. For example, the mice hybridoma cellstrains 8H5, 3C8, 10F7, 4D1, 3G4, and 2F2 were deposited in China Centerfor Typical Culture Collection (CCTCC, Wuhan University, Wuhan, China)on Jan. 17, 2006 with deposit numbers of CCTCC-C200607 (hybridoma cellstrain 8H5), CCTCC-C200605 (hybridoma cell strain 3C8), CCTCC-C200608(hybridoma cell strain 10F7), CCTCC-C200606 (hybridoma cell strain 4D1),CCTCC-C200604 (hybridoma cell strain 304) and CCTCC-C200424 (hybridomacell strain 2F2).

The present invention also provides monoclonal antibodies that block thebinding of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4, or 2F2 to thehemagglutinin of subtype H5 avian influenza virus. Such blockingmonoclonal antibodies may bind to the same epitopes on the hemagglutininthat are recognized by monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4,or 2F2. Alternatively, those blocking monoclonal antibodies may bind toepitopes that overlap sterically with the epitopes recognized bymonoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4, or 2F2. These blockingmonoclonal antibodies can reduce the binding of monoclonal antibodies8H5, 3C8, 10F7, 4D1, 3G4, or 2F2 to the hemagglutinin of subtype H5avian influenza virus by at least about 50%. Alternatively, they mayreduce binding by at least about 60%, preferably at least about 70%,more preferably at least about 75%, more preferably at least about 80%,more preferably at least about 85%, even more preferably at least about90%, even more preferably at least about 95%, most preferably at leastabout 99%.

The ability of a test monoclonal antibody to reduce the binding of aknown monoclonal antibody to the H5 hemagglutinin may be measured by aroutine competition assay such as that described in Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988). For example, such an assay could be performed bypre-coating a microtiter plate with antigens, incubating the pre-coatedplates with serial dilutions of the unlabeled test antibodies admixedwith a selected concentration of the labeled known antibodies, washingthe incubation mixture, and detecting and measuring the amount of theknown antibodies bound to the plates at the various dilutions of thetest antibodies. The stronger the test antibodies compete with the knownantibodies for binding to the antigens, the more the binding of theknown antibodies to the antigens would be reduced. Usually, the antigensare pre-coated on a 96-well plate, and the ability of unlabeledantibodies to block the binding of labeled antibodies is measured usingradioactive or enzyme labels.

Monoclonal antibodies may be generated by the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975). In the hybridomamethod, a mouse or other appropriate host animal is immunized by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in thehost animal by multiple subcutaneous or intraperitoneal injections. Itmay be useful to conjugate the immunizing agent to a protein known to beimmunogenic in the host animal being immunized, such as serum albumin,or soybean trypsin inhibitor. Examples of adjuvants which may beemployed include Freund's complete adjuvant and MPL-TDM. Afterimmunization, the host animal makes lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theantigen used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Desired lymphocytes are collected and fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59 103, Academic Press, 1996).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and MC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York, 1987).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107: 220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the cells may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103,Academic Press, 1996). Suitable culture media for this purpose include,for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cellsmay be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the sub clones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies of the invention may also be made by conventionalgenetic engineering methods. DNA molecules encoding the heavy and lightchains of the monoclonal antibodies may be isolated from the hybridomacells, for example through PCR using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies. Then the DNA molecules are insertedinto expression vectors. The expression vectors are transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein. The host cells are cultured under conditionssuitable for the expression of the antibodies.

The antibodies used bind to the H5 hemagglutinin with high specificityand affinity. The antibodies shall have low cross-reactivity with othersubtypes of hemagglutinin, preferably no cross-reactivity with othersubtypes of hemagglutinins, In one aspect, the invention providesantibodies that bind to H5 hemagglutinin with a K_(D) value of less than1×10⁻⁵M. Preferably, the K_(D) value is less than 1×10⁻⁶M. Morepreferably, the K_(D) value is less than 1×10⁻⁷M. Most preferably, theK_(D) value is less than 1×10⁻⁸M.

The antibodies of the invention may contain the conventional “Y” shapestructure comprised of two heavy chains and two light chains. Inaddition, the antibodies may also be the Fab fragment, the Fab′fragment, the F(ab)2 fragment or the Fv fragment, or another partialpiece of the conventional “Y” shaped structure that maintains bindingaffinity to the hemagglutinin. The binding affinity of the fragments tohemagglutinin may be higher or lower than that of the conventional “Y”shaped antibodies.

The antibody fragments may be generated via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys.Methods, 24:107-117, (1992) and Brennan et al., Science, 229:81 (1985)).Additionally, these fragments can also be produced directly byrecombinant host cells (reviewed in Hudson, Curr. Opin Immunol., 11:548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). Forexample, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163 167 (199)). In another embodiment, the F(ab′)₂ isformed using the leucine zipper GCN4 to promote assembly of the F(ab′)₂molecule. According to another approach, Fv, Fab or F(ab′)₂ fragmentscan be isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent toa person with ordinary skill in the art.

The present invention provides isolated nucleic acid molecules encodingantibodies or fragments thereof that specifically bind to H5hemagglutinin. Nucleic acid molecules encoding the antibodies can beisolated from hybridoma cells. The nucleic acid sequences of themolecules can be determined using routine techniques known to a personwith ordinary skill in the art. Nucleic acid molecules of the inventioncan also be prepared using conventional genetic engineering techniquesas well as chemical synthesis. In one aspect, the present inventionprovides an isolated nucleic acid molecule encoding the variable regionof the heavy chain of an anti-H5 (HA antibody or a portion of thenucleic acid molecule. In another aspect, the present invention providesan isolate nucleic acid molecule encoding the variable region of thelight chain of an anti-H5 (HA) antibody or a portion of the nucleic acidmolecule. In another aspect, the present invention provides an isolatednucleic acid molecule encoding the CDRs of the antibody heavy chain orlight chain variable regions.

Labeled Reagents

“Labeled reagent” refers to a substance comprising a detectable labelattached with a specific binding member (e.g., detection probe). Theattachment may be covalent or non-covalent binding, but the method ofattachment is not critical to the present invention. The label allowsthe label reagent to produce a detectable signal that is related to thepresence of analyte in the fluid sample. The specific binding membercomponent of the label reagent is selected to directly bind to theanalyte or to indirectly bind the analyte by means of an ancillaryspecific binding member, which is described in greater detailhereinafter. The label reagent can be incorporated into the test deviceat a site upstream from the capture zone, it can be combined with thefluid sample to form a fluid solution, it can be added to the testdevice separately from the test sample, or it can be pre-deposited orreversibly immobilized at the capture zone. In addition, the specificbinding member may be labeled before or during the performance of theassay by means of a suitable attachment method.

“Label” refers to any substance which is capable of producing a signalthat is detectable by visual or instrumental means. Various labelssuitable for use in the present invention include labels which producesignals through either chemical or physical means. Such labels caninclude enzymes and substrates, chromogens, catalysts, fluorescent orfluorescent like compounds and/or particles, magnetic compounds and/orparticles chemiluminescent compounds and or particles, and radioactivelabels. Other suitable labels include particulate labels such ascolloidal metallic particles such as gold, colloidal non-metallicparticles such as selenium or tellurium, dyed or colored particles suchas a dyed plastic or a stained microorganism, organic polymer latexparticles and liposomes, colored beads, polymer microcapsules, sacs,erythrocytes, erythrocyte ghosts, or other vesicles containing directlyvisible substances, and the like. Typically, a visually detectable labelis used as the label component of the label reagent thereby providingfor the direct visual or instrumental readout of the presence or amountof the analyte in the test sample without the need for additional signalproducing components at the detection sites.

Additional labels that can be utilized in the practice of the inventioninclude, chromophores, electrochemical moieties, enzymes, radioactivemoieties, phosphorescent groups, fluorescent moieties, chemiluminescentmoieties, or quantum dots, or more particularly, radiolabels,fluorophore-labels, quantum dot labels, chromophore-labels,enzyme-labels, affinity ligand-labels, electromagnetic spin labels,heavy atom labels, probes labeled with nanoparticle light scatteringlabels or other nanoparticles, fluorescein isothiocyanate (FITC), TRITC,rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7,Texas Red, Phar-Red, allophycocyanin (APC), epitope tags such as theFLAG or HA epitope, and enzyme tags such as alkaline phosphatase,horseradish peroxidase, F-galactosidase, alkaline phosphatase,˜-galactosidase, or acetylcholinesterase and hapten conjugates such asdigoxigenin or dinitrophenyl, or members of a binding pair that arecapable of forming complexes such as streptavidinlbiotin, avidinlbiotinor an antigen/antibody complex including, for example, rabbit IgG andanti-rabbit IgG; fluorophores such as umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin,green fluorescent protein, erythrosin, coumarin, methyl coumarin,pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue,dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin,fluorescent lanthanide complexes such as those including Europium andTerbium, Cy3, Cy5, molecular beacons and fluorescent derivativesthereof, a luminescent material such as luminol; light scattering orplasmon resonant materials such as gold or silver particles or quantumdots; or radioactive material include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,Tc99m, ³⁵S or ³H; or spherical shells, and probes labeled with any othersignal generating label known to those of skill in the art. For example,detectable molecules include but are not limited to fluorophores as wellas others known in the art as described, for example, in Principles ofFluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp,2nd edition (July 1999) and the 6ID Edition of the Molecular ProbesHandbook by Richard P. Hoagland.

A number of signal producing systems may be employed to achieve theobjects of the invention. The signal producing system generates a signalthat relates to the presence of an analyte (i.e., target molecule) in asample. The signal producing system may also include all of the reagentsrequired to produce a measurable signal. Other components of the signalproducing system may be included in a developer solution and can includesubstrates, enhancers, activators, chemiluminescent compounds,cofactors, inhibitors, scavengers, metal ions, specific bindingsubstances required for binding of signal generating substances, and thelike. Other components of the signal producing system may be coenzymes,substances that react with enzymic products, other enzymes andcatalysts, and the like. In some embodiments, the signal producingsystem provides a signal detectable by external means, by use ofelectromagnetic radiation, desirably by visual examination. Exemplarysignal-producing systems are described in U.S. Pat. No. 5,508,178.

In some embodiments, nucleic acid molecules can be linked to thedetection probe (e.g., antibody-linked oligonucleotides), whereby thenucleic acid functions as a label by utilizing nucleic acid labels. Forexample, a reagent solution or substrate comprised in a SCD can comprisedetection reagents—plurality of detection and capture specific bindingagents (“SBA”)—comprising a plurality of oligonucleotides functioning toprovide a detectable signal, whereby for a given subpopulation of SBAs(specific for a particular analyte), conjugated oligonucleotides arepre-stained with a different stain as compared to another subpopulationof antibodies (specific for a different analyte) are nucleic acid stainsthat bind nucleic acid molecules in a sequence independent manner.Examples include intercalating dyes such as phenanthridines andacridines (e.g., ethidium bromide, propidium iodide, hexidium iodide,dihydroethidium, ethidium homodimer-I and -2, ethidium mono azide, andACMA); some minor grove binders such as indoles and imidazoles (e.g.,Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); and miscellaneousnucleic acid stains such as acridine orange (also capable ofintercalating), 7-AAD, actinomycin 0, LDS751, and hydroxystilbamidine,All of the aforementioned nucleic acid stains are commercially availablefrom suppliers such as Molecular Probes, Inc. Still other examples ofnucleic acid stains include the following dyes from Molecular Probes:cyanine dyes such as SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-I,POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3,PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5,JO-PRO-I, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen,SYBR Gold, SYBR Green I, SYBR Green II, SYBR OX, SYTO-40, -41, -42, -43,-44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15,-14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64,-17, -59, -61, -62, -60, -63 (red). Other detectable markers includechemiluminescent and chromogenic molecules, optical or electron densitymarkers, etc.

As noted above in certain embodiments, labels comprise semiconductornanocrystals such as quantum dots (i.e., Qdots), described in U.S. Pat.No. 6,207,392. Qdots are commercially available from Quantum DotCorporation. The semiconductor nanocrystals useful in the practice ofthe invention include nanocrystals of Group II-VI semiconductors such asMgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS,ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well as mixedcompositions thereof; as well as nanocrystals of Group III-Vsemiconductors such as GaAs, InGaAs, InP, and InAs and mixedcompositions thereof. The use of Group IV semiconductors such asgermanium or silicon, or the use of organic semiconductors, may also befeasible under certain conditions. The semiconductor nanocrystals mayalso include alloys comprising two or more semiconductors selected fromthe group consisting of the above Group III-V compounds, Group II-VIcompounds, Group IV elements, and combinations of same.

In some embodiments, a fluorescent energy acceptor is linked as a labelto a detection probe. In one embodiment the fluorescent energy acceptormay be formed as a result of a compound that reacts with singlet oxygento form a fluorescent compound or a compound that can react with anauxiliary compound that is thereupon converted to a fluorescentcompound. Such auxiliary compounds can be comprised in buffers containedin an SCD and/or Test Device. In other embodiments, the fluorescentenergy acceptor may be incorporated as part of a compound that alsoincludes the chemiluminescer. For example, the fluorescent energyacceptor may include a metal chelate of a rare earth metal such as,e.g., europium, samarium, tellurium and the like. These materials areparticularly attractive because of their sharp band of luminescence.Furthermore, lanthanide labels, such as europium (III) provide foreffective and prolonged signal emission and are resistant to photobleaching, thereby allowing Test Devices containing processed/reactedsample to be set aside if necessary for a prolong period of time.

Long-lifetime fluorescent europium (III) chelate nanoparticles have beenshown to be applicable as labels in various heterogeneous andhomogeneous immunoassays. See, e.g., Huhtinen et al. Clin. Chem. 2004October; 50(10): 1935-6. Assay performance can be improved when theseintrinsically labeled nanoparticles are used in combination withtime-resolved fluorescence detection. In heterogeneous assays, thedynamic range of assays at low concentrations can be extended (1-3).Furthermore, the kinetic characteristics of assays can be improved byuse of detection antibody-coated high-specific-activity nanoparticlelabels instead of conventionally labeled detection antibodies (4). Inhomogeneous assays, europium (III) nanoparticles have been shown to beefficient donors in fluorescence resonance energy transfer, enablingsimple and rapid high throughput screening (5). Heterogeneous andhomogeneous nanoparticle-label-based assays can be run with varioussample matrixes, e.g., serum (3), heparin plasma (3), and nlUCUS (S.Huopalahti, A. Valanne, T. Soukka, R. Vainionpaa“, T. Lovgren, and H.Harma”, University of Turku, unpublished data).

In some embodiments, a label (e.g., fluorescent label) disclosed herein,is comprised as a nanoparticle label conjugated with biomolecules. Inother words, a nanoparticle can be utilized with a detection or captureprobe. For example, a europium(III)-labeled nanoparticle linked tomonoclonal antibodies or streptavidin (SA) to detect a particularanalyte in a sample can be utilized in practice of the present invention(e.g., nanoparticle-based immunoassay). The nanoparticles serve as asubstrate to which are attached the specific binding agents to theanalyte and either the detection (i.e., label) or capture moiety.

In one embodiment, a Test Device comprises different pRNAs eachpatterned based on a specific analyte, a complementary SCD comprises aplurality of capture antibody linked to cognate pRNAs to thoseimmobilized on the Test Device, and where said plurality comprisingdifferent subpopulation of antibodies specific for different analytes).Furthermore, the SCD reagent solution or substrate (e.g., lyophilizedsolid substrate) comprise detection probes, or a plurality of europium(III) labeled antibodies, consisting of the same subpopulations ofantibodies specific for different analytes. Additional lanthanide labelsthat can be practiced in the present invention are known in the art,such as disclosed in U.S. Pat. No. 7,101,667. See also, e.g., RichardsonF. S., “Terbium(III) and Europium(III) Ions as Luminescent probes andStains for Biomolecular Systems,” Chem. Rev., 82:541-552 (1982).

Therefore, depending on choice of labels, in some embodiments a signalis viewable by the unaided eye, while in other embodiments, a readerinstrument is utilized in the practice of the present invention.

Capture Moieties.

In some embodiments, one member of a pair of complementary capturemoieties will be bound to analyte-specific binding agent and the othermember is immobilized on a line or spot, respectively. As referred toherein, the terms “capture moiety” means a binding moiety that isspecific for a partner or complementary capture moiety (e.g., pRNAspecific for complementary pRNA, or avidin/streptavidin-bioin).

In some embodiments, pyranosyl RNA (pRNA) is provided as a series ofsynthetic oligonucleotides conjugated to monoclonal antibody(s) orpolyclonal antibodies in the assay system or method for detectingmultiple analytes from a single sample. pRNA contains D-ribose in apyranose form as opposed to furanose for RNA. Furthermore, the C-4′ andC-2′ phosphodiester linkage creates a rigid sugar-phosphate backboneresulting in rapid formation of highly stable pRNA duplexes. Rapidduplex formation contributes to non-specific cross-hybridization. Inadditional embodiments, an additional base, indole, is introduced todestabilize non-specific pRNA duplexes.

In some embodiments, pyranosyl RNA (pRNA) in a series ofoligonucleotides conjugated to monocloclonal antibodies. A complementarypRNA strand is bound to the nitrocellulose membrane to capture theappropriate monoclonal antibody-analyte complex at the test line.Additional embodiments are disclosed in related U.S. patent applicationSer. No. 11/677,559.

Where multiplexed (i.e., multianalyte) detection is desired, a pluralityof capture moieties is utilized, antibodies specific for one analyte(s)will comprise a member of one specific pair of complementary capturemoieties and antibodies that specifically bind a second and differentanalyte(s) will comprise a member of a second and different specificpair of complementary capture moieties, and so on. Thus, in oneembodiment, a plurality of different analyte(s) can be detected, wherethe cognate member of a pair of capture moieties is immobilized in adiscrete location on a test membrane comprised in the test implement.

In various embodiments, capture moieties are comprised of anoligonucleotide, avidin, streptavidin, pyranosyl RNA (pRNA),antigen-antibody binding pair selected for high affinity, aptamer or acombination thereof. In further embodiments, an oligonucleotide is DNAor RNA. Moreover, in some embodiments a combination of different capturemoieties are utilized in the same detection system of the invention. Forexample, a capture moiety pair for one specific analyte comprises anoligonucleotide pair, while a capture pair for a different analytecomprises a capture moiety pair comprising pRNA, or avidin orstreptavidin, etc. In one embodiment, all capture moieties are pRNAs,with multiple pairs of pRNA capture moiety and pRNA partner capturemoiety (e.g., one is conjugated to a specific binding agent and thecognate pRNA is immobilized on the lateral flow membrane).

In some embodiments, pRNA capture moities/capture moiety partners areselected from but not limited to the following pRNAs:

Name 4'-2' 102a10-3-NH2 TAGAACGAAG (SEQ ID NO: 1) 102b10-3-NH2CTTCGTTCTA (SEQ ID NO: 2) 119a10-1-NH2 TCAGTGGATG (SEQ ID NO: 3)119b10-1-NH2 CATCCACTGA (SEQ ID NO: 4) 3a10-1-NH2GTATTGCGAG (SEQ ID NO: 5) 3b10-1-NH2 CTCGCAATAC (SEQ ID NO: 6)102a8-2-NH2 AACGATTC 102b8-2-NH2 GAATCGTT 119a8-1-NH2 AGTGGATG11968-1-NH2 CATCCACT 3a8-1-NH2 GTATTGCG 3b8-1-NH2 CGCAATAC 4a9-InATGCDCTTC 4b8-In GAADGCAT 5a8 TGATGGAC 5b9-In GTCDCATCA 6a6 CAGTAG 6b6CTACTG 8a6 GACTCT 8b6 AGAGTC all oligos with 4′-C12 amino and 2′-hexanolgroups Note D = Indole, a neutral base that adds specificity to theoligonucleotide.

In other embodiments, a combination of different types of capturemoieties is utilized in devices and assays of the invention to detectmultiple analytes (e.g., plurality of capture probes and detectionprobes whereby each population of capture probe and detection probe isspecific for a single type of target analyte. Capture probes can beconfigured with one or more different capture moieties such as aptamers,pRNA or streptavidin, etc.)

In one embodiment, combinations capture probes utilizing pRNA capturemoieties are utilized to detect a plurality of different target analytes(e.g., 1, 2, 3, 4, 5 or 6 different viral antigens). For example, asillustrated in FIG. 4 a device of the invention is used to detect avirus at a higher sensitivity.

In some embodiments, a sample containing multiple analytes (e.g., 1, 2,3, 4, 5, or more) FIG. 5A is brought into contact with capture anddetection probes (e.g., FIG. 5A). Capture antibodies 60 are directed todifferent target analytes 54, 55 and all capture antibodies 60 specificfor a particular target have a pRNA capture moiety 56, 57 which iscapable of specifically binding to a immobilized capture moiety 58, 59.Therefore, where detection probes (e.g., antibody with detectable label)bind a specific target analyte, and capture probes bind a specifictarget analyte, the capture moieties 56, 57 provide a means to bind thetarget-detection probe-capture probe complex to a particular addressableline on the test device 50. In this way, a plurality of different targetanalytes can be detected (e.g., different pathogens, infectious agents,or types/subtypes of the same an infectious agent).

The devices and methods of the invention are configured to provideincreased specificity and sensitivity to a plurality of different targetanalytes.

In other embodiments, target analyte(s) can be polypeptides or peptidesassociated with a subject and not an infectious agent. In yet furtherembodiments, target analyte(s) can be associated with a cancer, tumor orneoplasm. As illustrated in FIG. 6, a target analyte can be detectedwith enhanced sensitivity (FIG. 6 illustrates a peptide, NTproBNPpeptide, detected utilizing pRNA capture moiety pairs to detect 10 pg/ml(lane 2), 100 pg/rnl (lane 3), 1000 pg/ml (lane 4), of NT-proBNPpeptide. The control lane that did not contain any peptide (lane 1) anddid not produce any detectable signal. In limited dilution assay usingNT-proBNP peptide, as shown in FIG. 6, the detection limit was 0.36pg/ml of peptide, which translates to 1.7 attamoles (10⁻¹⁸) of peptide.

Therefore, where a plurality of capture probes (e.g., antibody linked topRNA), each capture probe is linked to a capture moiety, for which acognate capture probe is immobilized in a predetermined location on atest strip comprised in a Test Device. For example, a plurality ofantibodies in an SCD is comprised of antibodies targeting differentinfluenza virus strains and/or subtypes, where said antibodies arecomprised of pairs of detection antibody-capture antibody and where thecapture antibody has a specific capture moiety. Further, each populationof antibodies in the plurality of antibodies is defined by theparticular target analyte to which the antibody binds. Thus, all captureantibodies directed to one specific target analyte will have the samecapture moiety, for which cognate/complementary capture moieties aredisposed in the Test Device.

In some embodiments, capture moieties are aptamer molecules that are canbe interchangeably utilized with a capture probe or as an immobilizedcapture moiety included in the Test Device axial flow membrane. Aptamersinclude nucleic acids that are identified from a candidate mixture ofnucleic acids. In a preferred embodiment, aptamers include nucleic acidsequences that are substantially homologous to the nucleic acid ligandsisolated by the SELEX method. Substantially homologous is meant a degreeof primary sequence homology in excess of 70%, most preferably in excessof 80%. The “SELEX” methodology, as used herein, involves thecombination of selected nucleic acid ligands, which interact with atarget analyte in a desired action, for example binding to a protein,with amplification of those selected nucleic acids. Optional iterativecycling of the selection/amplification steps allows selection of one ora small number of nucleic acids, which interact most strongly with thetarget antigen/biomarker from a pool, which contains a very large numberof nucleic acids. Cycling of the selection/amplification procedure iscontinued until a selected goal is achieved. The SELEX methodology isdescribed in the following U.S. patents and patent applications: U.S.patent application Ser. No. 07/536,428 and U.S. Pat. Nos. 5,475,096 and5,270,163.

Analytes

In one aspect of the invention, one or more analytes are detected usingthe device, system and methods of the invention. In various embodiments,one or more analytes include but are not limited to one or moremammalian cell, virus, bacteria, yeast, fungi, parasite, components ofthe preceding, nucleic acid, polypeptide, peptide, and combinationsthereof

In various embodiments, the analyte(s) detected are associated with aninfectious agent. An infectious agent can be any pathogen includingwithout any limitation bacteria, yeast, fungi, virus, eukaryoticparasites, etc. In some embodiments, the infectious agent is influenzavirus, parainfluenza virus, adenovirus, rhinovirus, coronavirus,hepatitis viruses A, B, C, D, E, etc, HIV, enterovirus, papillomavirus,coxsackievirus, herpes simplex virus, or Epstein-Barr virus. In otherembodiments, the infectious agent is Mycobacterium, Streptococcus,Salmonella, Shigella, Staphylcococcus, Neisseria, Clostridium, or E.coli. It will be apparent to one of skill in the art that thecompositions and methods of the invention are readily adaptable todifferent infectious agents, by utilizing a different panel of bindingagents (e.g., antibodies) that are specific for type(s) or subtype(s) ofan infectious agent(s).

Usually the general type of an infectious agent can be the genus type ofan infectious agent or any primary or first instance typing oridentification of an infectious agent. A subtype of an infectious agentcan be the species or strain type of an infectious agent or anysecondary or subsequent typing of an infectious agent. According to thepresent invention, identification of the general type or subtype of aninfectious agent can be carried out via various suitable test set ups.For example, identification of the general type of an infectious agentcan include one or more screening tests for 1) a specific general typeof an infectious agent, 2) certain desired or selected general types ofan infectious agent, or 3) all or substantially all relevant generaltypes of an infectious agent, or a combination thereof. Similarlyidentification of the subtype of an infectious agent can include one ormore screening tests for 1) one or more specific subtypes of aninfectious agent, 2) one or more specific subtypes of a particulargeneral type of an infectious agent, 3) one or more specific subtypes ofan infectious agent selected based on additional information associatedwith the subject being tested, e.g., one or more suspected or expectedsubtypes for a particular population or geographic location or 4) one ormore potentially pandemic or epidemic subtypes of an infectious agentthat is identical to or associated with the infectious agent tested forthe general type, or a combination thereof.

In particular, the general type of an influenza virus can be any typedesignated based on antigenic characteristics of the nucleoprotein andmatrix protein antigens, e.g., type A, B, or C influenza virus, whereasthe subtype can be one or more subdivided types of an influenza virus onthe basis of an antigen, e.g. one or more subtypes of influenza type Aor type B virus characterized by a surface antigen such as hemagglutinin(H) or neuraminidase (N).

In one embodiment, identification of the general type of influenza virusincludes screening for type A, type B, and/or type C influenza viruswhereas identification of the subtype of influenza virus, e.g., type Avirus includes screening for one or more expected subtypes of type A,e.g., subtypes expected to be present in the population at the time oftesting, and optionally one or more suspected subtypes, e.g., subtypesunder surveillance for an outbreak such as epidemic or pandemicoutbreak. In another embodiment, identification of the general type ofinfluenza virus includes screening for type A and type B influenza viruswhereas identification of the subtype of influenza virus, e.g., type Avirus includes screening for one or more subtypes used for theproduction of the influenza vaccine, e.g., current vaccine subtypes(s)or strain(s) for the testing season including subtypes and/or strainsexpected to be in circulation during the next influenza season. In yetanother embodiment, identification of the general type of influenzavirus includes screening for type A and type B influenza virus whereasidentification of the subtype of influenza virus, e.g., type A includesscreening for one or more subtype(s) or strain(s) used for theproduction of the influenza vaccine and one or more subtype(s) orstrain(s) suspected for the cause of a pandemic outbreak, e.g., one ormore avian subtype(s) or strain(s) such as H5N1 or the derivativesthereof.

In one embodiment, the methods and compositions of the invention can beutilized in assays to detect E. coli 0157 (a very dangerous, often fatalinfectious strain) in the presence of other enteric or infectivestrains. Another example would be in testing patients for influenzainfection, where mutation or variation of the strains within subtypes isknown to occur and some forms of influenza are far more pathogenic thanothers. A further example is detection of different types of HIV, forexample HIV-1 and HIV-2.

In one embodiment, the methods and apparatus of the invention areutilized to detect or identify an influenza type A subtype and/orinfluenza type B.

Influenza virus belongs to the genus orthomyxovirus in the family ofOrthomyxoviridae. ssRNA enveloped viruses with a helical symmetry.Enveloped particles 80-120 nm in diameter. The RNA is closely associatedwith the nucleoprotein (NP) to form a helical structure. The genome issegmented, with 8 RNA fragments (7 for influenzaC). There are 4principle antigens present, the hemagglutinin (H), neuraminidase (N),nucleoprotein (NP), and the matrix (M) proteins. The NP is atype-specific antigen which occurs in 3 forms, A, Band C, which providesthe basis for the classification of human and non-human influenzaviruses. The matrix protein (Mprotein) surrounds the nucleocapsid andmakes up 35-45% of the particle mass. Furthermore, 2 surfaceglycoproteins are seen on the surface as rod-shaped projections. Thehaemagglutinin (H) is made up of 2 subunits, HI and H2. Haemagglutininmediates the attachment of the virus to the cellular receptor.Neuraminidase molecules are present in lesser quantities in theenvelope. The antigenic differences of the hemagglutinin and theneuraminidase antigens of influenza A viruses provide the basis of theirclassification into subtypes. e.g., AlHong Kong/1168 (H3N2) signifies aninfluenza A virus isolated from a patient in 1968, and of subtype H3N2.

In various embodiments, the methods and apparatus of the invention aredirected to detecting or identifying influenza virus type A which isdefined by HxNy where x is 1-9 and y is 1-16, or any combination of xythereof. For example, in one embodiment, the methods and apparatus ofthe invention is utilized to detect influenza A subtype HIN5. Thus, aplurality of detection probes and capture probes targeting differentsubtypes of influenza virus are disposed in an seD of the invention. Inone embodiment, the assay is utilized to detect Influenza A (withsubtypes H1/H3, and a pandemic subtype H5) and Influenza B.

In various embodiments, methods and apparatus of the invention candetect one or more different infectious agents. For example, a samplingimplement can comprise a plurality of different antibodies, whereinmultiple subgroups of antibodies are present, whereby each subgroup ofantibodies specifically binds a different infectious agent. For example,a plurality of antibodies can comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10subgroups, wherein each subgroup of antibodies in the plurality ofantibodies specifically binds a different infectious agent. In someembodiments, methods and apparatus of the invention detect a pandemicand non-pandemic infectious agent. In one embodiment, the pandemic andnon-pandemic infectious agents are influenza virus.

The explosive nature of epidemic influenza and the specific clinicalfeatures of this disease have given reliable epidemiological records ofthis infection since the beginning of the nineteenth century. Severalepidemics were recorded during the nineteenth century but the firstpandemic was not accurately recorded until 1889-92. A second pandemic,probably originating in Europe, occurred in 1918-19, and is known asSpanish Influenza, which was responsible for 20-25 million deaths,principally in young adults.

Pandemics continued to occur regularly after the Spanish influenza, in1932-33, 1947-48, 1957 and 1968. The next pandemic is thought to beoverdue. These latter pandemics resembled the pandemic of 1890,affecting millions of people with a mild URTI and a small number ofdeaths. The HINI (swine) viruses probably appeared in 1918 and continuedto circulate until 1957, at which time they were supplanted by the H2N2(Asian) viruses. The H2N2 viruses were prevalent until 1968, when H3N2(Hong Kong) strains appeared. The H1N1 virus reappeared in 1977 and didnot replace the H3N2 subtype and both subtypes continued to cocirculate.Therefore, it is imperative that subjects are screened in an effectiveand accurate manner to determine with what strain and/or subtype anindividual is infected. Furthermore, in some circumstances such samplecollection and processing will necessarily occur in a point-of-caresetting (e.g., in the field, without large numbers of subjects to sampleand process, and with limited man power to effect such sampling).

As such, in one embodiment, the methods and apparatus of the inventionare utilized in processing a large number of samples, in a point-of-caresetting, where test results may be visualized (i.e., read) some periodof time after the test is complete. For example, the period of time canbe 30 minutes, 1 hour, 1.5 hour, 2 hours, 2.5 hours, 3 hours, 4 hours or5 hours. In some embodiments, methods and apparatus in conjunction withthe reagents disclosed herein provide high sensitivity and specificitywhere the fluorescent result can be read with very similar results overa long period of time. Thus, in some embodiments biological samples canbe collected and processed, but set aside to be read a significant timelater, which is greatly advantageous in point-of-care settings or wherea large number of samples are collected with limited manpower or time tofurther process samples.

In various embodiments, analytes detected are one or more biomarkerswhich are indicative of a disease, condition or predisposition to adisease or condition. Examples of such analytes include but are notlimited to tumor antigens, peptides indicative of stroke, peptidesindicative of hear failure heart failure, peptides indicative ofpreclampsia, eclampsia, liver damage or disease, kidney damage ordisease, heart damage or disease, brain damage or disease, or anycombination thereof.

In various embodiments, detection of markers (also biomarkers) can bebefore during or after treatment, and as such, detection of one or moremarkers is used to assess the efficacy of or a subject's response to atreatment regimen.

In various embodiments, devices and methods of the invention areconfigured to detect one or more markers (e.g., peptides or antigens)associated with any condition or disease. For example, one or moremarkers can be detected which are associated with any disease orcondition, including but not limited to a cancer or tumor, a heartcondition, damage or disease, a brain condition, liver condition, damageor disease, kidney condition, damage or disease, or a combinationthereof. In further embodiments, detection of one or more markers isbefore, during or after treatment of a subject with a therapeutic agent.Thus, a treatment regimen for a subject or population of subjects can beassessed to determine the efficacy of a therapeutic regimen. In yetother embodiments, markers associated with one or more adverse ornegative effects can be detected to determine the incidence or severityof adverse effects. In yet other embodiments, one or more markers aredetected to diagnose a disease or condition.

Example of detection markers can be tumor antigens associated withcancer. Examples of such tumor cell components include, but are notlimited to, epidermal growth factor receptor (EGFR, ErbB-1, HER1),ErbB-2 (HER2/neu), ErbB-3HER3, ErbB-4/HER4, EGFR ligand family,insulin-like growth factor receptor (IGFR) family, IGF-binding proteins(IGFBPs), IGFR ligand family, platelet derived growth factor receptor(PDGFR) family, PDGFR ligand family, fibroblast growth factor receptor(FGFR) family, FGFR ligand family, vascular endothelial growth factorreceptor (VEGFR) family, VEGF family, HGF receptor family, TRK receptorfamily, ephrin (EPH) receptor family, AXL receptor family, leukocytetyrosine kinase (LTK) receptor family, TIE receptor family, angiopoietin1,2, receptor tyrosine kinase-like orphan receptor (ROR) receptorfamily, discoidin domain receptor (DDR) family, RET receptor family, KLGreceptor family, RYK receptor family, MuSK receptor family, Transforminggrowth factor beta (TGF-β), e.g., three isoforms called TGF-β1, TGF -β2and TGF-β3, Cytokine receptors, Class I (hematopoietin family) and ClassII (interferon/IL-10 family) receptors, tumor necrosis factor (TNF)receptor superfamily (TNFRSF), death receptor family(Apo2L/TRAIL-Receptors, CD95/Fas), cancer-testis (CT) antigens,lineage-specific antigens, differentiation antigens, alpha-actinin-4,ARTC1, breakpoint cluster region-Abelson (Bcr-abl) fusion products,B-RAF, caspase-5 (CASP-5), caspase-S(CASP-8), β-catenin (CTNNB1), celldivision cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A,COA-1, dek-can fusion protein, EFTUD-2, Elongation factor 2 (ELF2), Etsvariant gene 6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusionprotein, fibronectin (FN), GPNMB, low density lipid receptor/GDP-Lfucose: β-Dgalactose 2-a-Lfucosyltransferase (LDLR/FUT) fusion protein,HLA-A2. arginine to isoleucine exchange at residue 170 of the a helix ofthe α2-domain in the HLA-A2 gene (HLA-A *201-R170I), HLA-A11, heat shockprotein 70-2 mutated (HSP70-2M), KIAA0205, MART2, melanoma ubiquitousmutated 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP),neo-PAP, Myosin class I, NFYC, OGT, OS-9, pml-RARalpha fusion protein,PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1,SYT-SSX1 or-SSX2 fusion protein, Triosephosphate Isomerase, BAGE,BAGE-1, BAGE-2,3,4,5, GAGE-1,2,3,4,5,6,7,8, GnT-V (aberrantN-acetylglucosaminyl transferase V, MGAT5), HERV-K-MEL, KK-LC, KM-HN-I, LAGE,LAGE-1, CTL-recognized antigen on melanoma (CAMEL), MAGE-A1 (MAGE-1),MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGEA6, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGEB5, MAGE-B6, MAGE-C1,MAGE-C2, mucin I (MUC1), MART-1/Melan-A (MLANA), gp100, gp100/Pme117(SILV), tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2,SAGE, Sp17, SSX-1,2,3,4, TRP2-INT2, carcino-embryonic antigen (CEA),Kallikrein 4, mammaglobin-A, OA1, prostate specific antigen (PSA),TRP1/gp75, TRP-2, adipophilin, interferon inducible protein absent inmelanoma 2 (AIM-2), BING-4, CPSF, cyclin D1, epithelial cell adhesionmolecule (Ep-CAM), EphA3, fibroblast growth factor-5 (FGF-5),glycoprotein 250 (gp250), EGFR (ERBB1), HER-2/neu (ERBB2), interleukin13 receptor a chain (IL13Ralpha2), IL-6 receptor, intestinal carboxylesterase (iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUC1, p53(TP53), PBF, PRAME, PSMA, RAGE-I, RNF43, RU2AS, SOX10, STEAP1, survivin(BIRC5), human telomerase reverse transcriptase (hTERT), telomerase,Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1,CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA661, LDHC,MORC, SGY-1, SP011, TPX1, NY-SAR-35, FTHL17, NXF2, TDRD1, TEX15, FATE,TPTE, immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors(ER), androgen receptors (AR), CD40, CD30, CD20, CD19, CD33, cancerantigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA19-9), β-human chorionic gonadotropin, 13-2 microglobulin, squamous cellcarcinoma antigen, neuron-specific enolase, heat shock protein gp96,GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinomaantigen recognized by T cells 4 (ART-4), carcinoembryogenic antigenpeptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2),cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), Humanpapilloma virus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsidantigens, others), Epstein-Barr virus (EBV) proteins (EBV latentmembrane proteins—LMP1, LMP2; others), Hepatitis B or C virus proteins,and HIV proteins.

In other embodiments, detection markers peptides or cell componentsassociated with a disease, condition, or organ damage. Examples of suchmarkers include, but are not limited to those disclosed in U.S. Pat. No.7,361,473, such as brain natriuretic peptide (BNP), NT-proBNP, proBNP,CNP, and ANP, cardiac troponins, C reactive protein, lipolipoprotein A,NCA 50/90, 36P6D5, glutathione-S-transferase PI (GSTP1), lipoproterinassociated cholesterols, HLLRCR-1, KIR4.1, brain associated humanglutamine synthetase or matrix metalloproteins, and variants thereof,including but not limited to markers disclosed in U.S. Pat. Nos.5,605,894, 6,709,818, 6,028,055, 7,223,542, 7,332,569, 7,348,149,7,262,290, or U.S. Pat. No. 7,070,945 and W0/2005/072055.

Work Flow:

In general, a sample is processed using an SCD of the invention whichprepares a sample for application to a TD and reading a result via aReader, as illustrated in FIG. 7. For example, in some embodiments, asample is obtained or added to a SCD FIG. 7, panel A and one or moreanalytes are contacted with reagents present in the SCD (e.g., detectionand capture probes, and/or extraction reagents). The sample is thenprocessed through the SCD to the TD as illustrated in FIG. 7, panel B,and read on a optical reader FIG. 7, panel C. For example, the samplingimplement can be used to swab a cell, tissue or liquid sample from asubject FIG. 7, panel A where an extraction buffer (supra) is released.The SCD is then placed into a TD as illustrated in FIG. 7, panel B, anda bulb on the SCD is squeezed to transfer fluid from the SCD to the TD.Alternatively a buffer present in the TD (e.g., wash or running bufferwith or without additional reagents, dyes, labels) is released once thesnap button FIG. 2 on the TD is pressed. Subsequently or a substantialtime thereafter the TD is inserted into an optical reader to detectpresence of a detectable signal at one or more defined lines on the TD.In some embodiments, the TD is read in about 5, 10, 15, 20, 30, 40, 50or 60 minutes. However, if necessary (e.g., operator has multiple TDs toprocess) the TD is configured to provide accurate reads from severalhours (e.g., 1,2,3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 hours) or to a few days after running the sample(e.g., 1,2,3,4,5,6 or 7 days). As discussed in the

In one embodiment, after the health care worker collects a specimen asampling implement (e.g., swab) is inserted part way into the plastictube and the handle of the swab is broken off and discarded. The handleof the SCD with the extraction reagents is affixed to the tube, thevalve broken, the bulb squeezed to move the fluid to the swab tip (e.g.,FIG. 7, panel A).

In one embodiment, a sample is collected from a subject via a samplingimplement and placed back into the cylinder housing of the SCD device(e.g., FIG. 7). The SCD can first be inserted into a Test Device, orprior to insertion into a Test Device, a solution contained in the uppersealed chamber of the SCD is released to effect washing the sample andsolution into a mixture downwards into a reaction chamber. In thereaction chamber is disposed either liquid or solid reagents comprisingdetection and capture probes that target one or more different analytesas disclosed herein, thereby forming a complex of analyte bound todetection and capture probe. The sample is then expelled from the SCDinto a Test Device through an aperture that seals the contact betweenthe SeD and the Test Device from the outside environment (e.g.,preventing any spillage, aerosol or contamination). The sample mixturecan flow as a result of gravity or the force of air pressure produced bysqueezing the SCD (e.g., upper sealed chamber), into a Test Device. Thesample is driven by capillary force and/or by wash buffer comprised inthe Test Device so as to allow any analyte-probe complex to pass throughthe lateral flow membrane contained in the Test Device. Capture probesand complementary immobilized capture moieties bind or hybridize to eachother in predetermined lines or spots on the lateral flow membrane,whereby detection probes (via conjugate labels contained thereon) willprovide a detectable signal which can subsequently be read to determinewhich analytes were present in the sample processed.

In one embodiment, Test Devices with samples processed thereon, can beset aside for time periods of about 1, 2, 3, 4, 5, 6 or 8 hours beforereading the results, and yet provide results as accurately as if read in15 or 20 minutes after processing. Thus, the signals produced are stablefor long periods of time so that reading the results may occur at asignificantly later time after the tests are actually performed. This isa great improvement for point of care diagnostics, where in the fieldconditions often present limited resources in manpower and time, andwhere the test setting can be in remote regions that are not easily orquickly accessed.

For example, the cap is removed and the SCD is attached to the TD. Thebulb is squeezed again. The extracted sample dissolves the reagent beadand the mixture is transferred to the wicking pad of the TD. The buttonon the TD is depressed starting the flow of wash buffer.

The TD is placed into the reader. The operator enters their ID into thereader and depresses the “start” button. No other operator interactionwith the reader is required other than to remove the TD from the reader.The reader will automatically display and print the result and with theaddition of communication connectivity capability the system will makethe result available to be downloaded.

EXAMPLES Example 1 Detecting Different Influenza Types/Subtypes

Assay was performed on the following influenza strains according to theguidelines set by the Center for Disease Control; New Caiedonia/20/99H1N1 influenza A, Hawaii/15/01 H1N1 influenza A, New York/55/04 H3N2influenza A, Wisconsin/67/05/H3N2 influenza A, Florida/07/06 influenzaB, Ohio/15/01 influenza B, an H5 influenza A, and an H5 influenza V.Each strain was diluted to 1:100, 1:750, 1:1000, 1:7,500, or 1:10,000for testing the sensitivity of the assay system. Normalized test resultswere presented in Table 1. showing test results of on various influenzastrains, demonstrating the assay can clearly distinguish between Type A,Type B, H1/H3 (detected together), and H5.

TABLE 1 Virus Dilution Control H5 H1/H3 Flu B FluA AlNew 1:100 16.390.00 6.73 0.05 40.38 Caledonia/20/99 1:1000 17.39 0.00 0.61 0.25 4.76(H1N1) 1:100 1:7500 16.28 0.00 0.00 0.08 0.92 TCID50 titer 4.6End 1:750014.44 0.00 0.20 0.28 0.94 point titer: −600 1:10000 15.48 0.00 0.00 0.001.02 1:100 15.53 0.00 0.63 0.23 38.23 A/Hawaii/15/2001 1:750 13.73 0.000.41 0.00 9.41 (H1N1) 1:100 1:1000 16.12 0.00 0.00 0.00 7.56 TCID50titer 4.7End 1:7500 15.48 0.00 0.00 0.00 1.13 point titer: −700 1:1000015.49 0.02 0.13 0.00 0.97 1:1000 23.10 0.00 1.17 0.14 11.37 A/New 1:750015.02 0.00 0.26 0.12 1.61 York/55/2004(H3N2) 1:7500 15.53 0.00 0.16 0.301.86 1:100 TCID50 titer 1:10000 16.57 0.00 0.00 0.22 1.48 3.5 End pointtiter: 1:10000 16.67 0.00 0.11 0.00 1.88 −10 1:100 15.53 0.10 2.76 0.1756.34 A/Wisconsin/67/2005 1:750 15.67 0.00 0.76 0.00 11.12 1:1000 16.580.00 0.47 0.00 9.09 (H3N2) 1:100 1:7500 15.33 0.00 0.09 0.02 1.42 TCID50titer 4.6 1:10000 20.98 0.00 0.00 0.00 1.33 End point titer: −300 1:10016.08 0.00 0.16 12.81 0.36 B/Florida/07/2006 1:1000 17.11 0.11 0.33 1.340.24 1:100 TCID50 titer 1:7500 16.95 0.05 0.17 0.19 0.25 4.5 End pointtiter: 1:7500 15.62 −0.30 0.26 0.14 0.00 −250 1:10000 16.49 0.00 0.00−0.05 0.24 1:1000 22.09 0.00 0.00 11.26 0.42 B/Ohio/15/2001 1:7500 18.660.00 0.08 2.35 0.36 1:100 TCID50 titer 1:10000 16.99 0.00 0.00 1.83 0.303.5 1:10000 15.88 0.00 0.00 2.16 0.44 End point titer: −10 1:75000 16.190.00 0.60 0.51 0.58 H5A 1:100 13.32 1.17 0.35 0.23 79.50 1:100 TCID50titer 1:750 15.13 0.00 0.00 0.04 33.89 5.7 1:1000 15.01 0.00 0.14 0.0029.15 End point titer: −700 1:7500 15.73 0.00 0.07 0.00 4.78 H5V 1:1000015.57 0.00 0.00 −0.01 3.58 1:100 TCID50 titer 1:100 14.20 1.40 0.40 0.2172.24 5.7 1:750 15.47 0.00 0.00 −0.04 22.37 End point titer: −700 1:100015.67 0.00 0.00 −0.11 19.89 1:7500 15.89 0.00 0.04 0.32 2.86 No VirusControl 1 1:10000 16.08 0.00 0.00 0.00 2.86 No Virus Control 2 16.58−1.08 0.14 0.00 0.28 No Virus Control 3 15.59 0.00 0.00 0.00 0.18 NoVirus Control 4 14.43 0.38 0.00 0.40 0.33

Example 2 Assay Specificity

To test whether the POC assay system can stringently identify influenzastrains in samples including non-S influenza bacteria and virus, thefollowing microorganisms were tested for the possibility of giving falsepositive signals; K. oxytoca, P. aeruginosa, P. mirabilis, S. aureus, S.aureus-303802, S. maltophilia, S. marcescens, S. simulans,Cytomegalovirus culture fluid, Herpes simplex virus culture fluid,Epstine B virus culture fluid, Parainfluenza culture, Coxsackievirusculture, Echovirus culture, Coronavirus culture Rous sarcoma virusculture, and Adenovirus culture. Normalized results were presented inTable 2, demonstrating assay specificity with respect to crossreactivity with non-influenza bacteria and virus.

TABLE 2 Sample Name Control H5 H1/H3 Flu B FluA Call TBS/Casein/NaN3-117.39 0.09 0.22 0 .04 0.00 Neg TBS/Casein/NaN3-2 17.87 0.03 0.33 0.20−0.03 Neg H. influenzae 18.30 0.00 0.03 0.11 0.00 Neg K. oxvtoca 17.160.34 0.59 0.41 0.38 Neg P. aerucinosa 13.42 0.21 0.48 0.37 0.32 Neg P.mirabilis 18.26 0.00 0.34 0.25 0.33 Neg S. aureus 17.85 0.17 0.19 0.000.03 Neg S. aureus-303802 14.24 0.00 0.75 0.00 0.95 Neg S. maltoohilia17.36 0.12 0.01 0.01 0.22 Neg S. marcescens 16.34 0.00 0.20 0.09 0.20Neg S. simulans 17.63 0.00 0.17 0.13 0.35 Neg CMV Culture 17.35 0.000.04 0.02 0.02 Neg Fluid HSV Culture Fluid 5.39 0.10 0.20 0.08 0.08 NegEBV Culture Fluid 17.69 0.33 0.44 0.29 0.23 Neg Parainfluenza 18.26 0.000.02 0.18 −0.02 Neg Culture Coxsackievirus 17.34 0.00 0.37 0.31 0.51 NegCulture Echovirus Culture 15.70 0.32 0.38 0.44 0.02 Neg Coronavirus17.40 0.15 0.02 0.10 0.06 Neg Culture RSV Culture 14.98 0.51 0.23 0.140.05 Neg Adenovirus 13.98 0.00 0.00 0.22 0.12 Neg Culture

Example 3 Drug Interference

To test whether the POC assay system can identify influenza virus fromsamples containing drugs commonly taken for influenza infection, thefollowing drugs were added in the sample containing HINI influenza A infollowing specified doses; 10 mg/ml of, Tylenol, 10 mg/ml of BayerAspirin, 10 mg/ml of Advil, 5 mg/ml of Bromphen, 5 mg/ml of ChlorTrimeton, 2.5 mg/ml of Symmetrel, 1 mg/ml of Ventolin, 10 mg/ml ofTussin, 10 mg/ml of Echinacea, 5 mg/ml of Galphimia G, 5 mg/ml ofHistamine HC, 5 mg/ml of Oscillococcinum, 10 mg/ml of Sucrets Menthol.Normalized test results were presented in Table 3, showing test resultsof the assay system on interference with common drugs that might betaken during an influenza infection, demonstrating unhindered assayperformance in the presence of influenza drugs in test sample.

TABLE 3 Final Conc. Virus IntSub Present (mg/mL) Sample Control H5 H1/H3B A +V Positive Control 1st 14.83 0.05 66.28 0.2 68.52 +V PositiveControl 14.14 0.09 72.28 0.02 59.89 2nd +V 10 Tvlenol 12.59 0.35 50.221.08 57.44 +V 10 Bayer 14.95 0.00 8.64 0.35 52.17 +V 10 Advil (retest)13.66 0.26 8.58 0.08 49.64 +V 5 Bromohen 10.04 0.54 30.01 0.15 45.79 +V5 Chlor Trimeton 9.50 0.08 23.02 0.10 47.50 +V 2.5 Svmmetrel 8.58 0.0044.93 0.00 53.20 +V 1 Ventolin 8.31 0.12 64.67 0.00 59.37 +V 10 Tussin8.86 0.00 49.02 0.22 51.68 +V 10 Echinacea 17.05 0.00 64.78 0.00 5.41 +V5 GalphimiaG 9.34 0.10 53.30 0.17 40.94 +V 5 Hist HC 9.13 0.14 44.050.00 42.03 +V 5 Oscillococcinum 11.69 0.00 80.79 0.00 50.07 +V 10Sucrets Menthol 13.00 0.00 33.90 0.15 53.55 +V 10 Benadryl 0.38 0.200.53 0.30 3.03 +V 1 Benadryl 9.23 0.26 52.27 0.08 63.32 +V 0.1 Benadryl11.20 0.13 40.33 0.00 51.65 +V 0.01 Benadryl 13.48 0.08 42.48 0.00 45.24−V Negative control 16.95 0.12 0.67 0.32 0.84 1^(st) −V Negative control13.32 0.00 0.16 0.07 0.06 2^(nd) −V 10 Tylenol 17.51 0.50 0.89 0.29 0.90−V 10 Bayer 8.86 0.11 0.06 0.07 0.02 −V 10 Advil (retest) 20.89 0.000.45 0.48 0.71 −V 5 Bromohen 12.26 0.21 0.26 0.10 0.04 −V 5 ChlorTrimeton 12.95 0.00 0.80 0.69 0.18 −V 205 Svmmetrel 12.04 0.22 0.19 0.070.04 −V 1 Ventolin 14.29 0.00 0.22 0.25 0.40 −V 10 Tussin 18.58 0.000.14 0.20 0.02 −V 10 Echinacea 15.93 0.00 0.21 0.20 0.00 −V 5 GalphimiaG18.10 0.00 0.11 0.04 0.10 −V 5 Hist HC 6.93 0.03 0.18 0.01 0.08 −V 5Oscillococcinum 17.76 0.00 0.39 0.00 0.26 −V 10 Sucrets Menthol 16.750.00 0.36 0.06 0.17 −V 10 Benadryl 1.09 2.91 3.15 2.24 2.07 −V 1Benadryl 12.59 0.18 0.29 0.00 0.08

Example 4 Influenza Sensitivity and Specificity

To test whether the POC assay system can correctly type live influenzavirus collected from patient, live Hong Kong influenza virus sampleswere tested. Table 4 shows test results of the assay system on live HSHong Kong influenza strain, demonstrating the feasibility and markedimprovement in detection capacity over a prior art. Also, Table 5 showsthe assay system is far more sensitive than prior art product indetecting Type A influenza.

TABLE 4 Titer measured H5 Strains by a represent prior art H5's Global(type A spread Virus Oil Control H5 H1/H3 TypeB Type A Call only)CklMalang/BB 1:10 8.97 39.33 0.80 0.00 45.46 H5 & A Pos Pos VET/4I04(Y1)1:100 9.65 4.70 0.35 0.21 58.60 H5 &A Pos Pos 1:1,000 11.10 0.95 0.600.23 26.30 APes WkPos 1:10,000 13.00 0.11 0.15 0.00 2.46 APos Neg1:100,000 11.20 0.13 0.13 0.21 1.20 APos neg ION 5105 1:10 8.88 34.190.68 0.10 36.67 H5 &A Pos Pos 1:100 8.95 3.42 0.19 0.00 57.72 H5 &A PosPos 1:1,000 8.72 0.59 0.58 0.51 s 38.84 APos Pos 1:10,000 19.24 0.530.17 0.68 1.41 APos Neg 1:100,000 11.63 0.34 0.49 0.10 1.18 APos NegCkIGXI2439/04 1:10 9.36 66.38 1.43 0.10 38.94 H5 H1/3 & Pos APos 1:1008.90 14.56 0.29 0.10 48.85 H5 &A Pos Pos 1:1,000 9.75 1.82 0.20 0.1454.22 H5 &A Pos Pos 1:10,000 11.62 0.57 0.51 0.41 9.96 APos Neg1:100,000 11.58 0.24 0.16 0.20 1.97 APos Neg BhGs/QH/15/05 1:10 15.2731.86 0.56 0.04 63.35 H5 &APos Pos 1:10 15.27 31.86 0.56 0.04 63.35 H5H1/3 & Pos APos 1:1,000 14.80 1.01 0.73 0.21 9.51 H5 &A Pos Pos 1:10.00010.45 0.44 0.47 0.17 1.03 APos Neg 1:100,000 11.22 0.15 0.12 0.11 0.37Neg Neg Little 1:10 5.00 6.21 0.40 0.09 46.84 H5 &A Pes PosEgreUHKI718/06 1:100 10.91 1.45 0.10 0.07 72.28 H5 &A Pos Pos 1:1,00011.82 0.19 0.16 0.11 30.92 A Pos Wk Pos 1:10,000 7.89 0.46 0.44 0.286.42 APos Neg 1:100,000 6.77 0.61 0.80 0.50 1.05 APos Neg GslYN/5539/2051:10 7.11 1.21 0.49 0.43 41.65 H5 &A Pos Pos 1:100 8.68 0.52 0.23 0.1242.22 A Pos Pos 1:1,000 11.43 0.55 0.68 0.40 48.22 A Pos Pos 1:10,00011.05 0.30 0.44 0.24 14.67 A Pos Neg 1:100,000 8.91 0.30 0.26 0.13 0.99Neg Neg DkNNM/N-XXIO4 1:10 9.79 5.75 0.16 0.00 47.18 H5 &A Pos Pos 1:10013.81 0.83 0.82 0.50 38.98 A Pos Pos 1:1,000 12.37 0.15 0.21 0.10 10.27A Pos Neg 1:10,000 15.09 0.20 0.44 0.10 2.66 A Pos Neg 1:100,000 13.480.00 0.00 0.00 0.73 Neg Neg MDkJJK2295/2005 1:10 8.47 8.84 0.41 0.1637.48 H5 & A Pos Pos 1:100 13.70 1.12 0.90 0.67 65.85 H5 & A Pos Pos1:1,000 11.29 0.40 0.41 0.29 54.92 A Pos Pos 1:10,000 13.41 0.22 0.240.15 16.47 APos WkPos 1:100,000 11.62 0.27 0.31 0.11 4.62 A Pos NegDKIST/4231/03 1:10 6.15 34.04 0.61 0.00 48.24 H5 & A Pos Pos 1:100 10.944.51 0.53 0.31 43.05 H5 & A Pos Pos 1:1,000 11.33 0.49 0.08 0.06 14.37 APos Neg 1:10,000 11.18 0.00 0.13 −0.05 2.01 A Pos Neg 1:100,000 12.660.37 0.43 0.19 0.51 Neg Neg Japaneese 1:10 8.92 4.34 0.46 0.20 52.81 H5&A Pos Pos White 1:100 10.30 0.69 0.54 0.32 28.36 A Pos PosEyeJHKI1038/06 1:1,000 10.98 0.00 0.05 0.00 8.98 A Pos Pos 1:10,00011.37 −0.03 0.21 0.00 0.98 Neg Neg 1:100,000 9.16 0.32 0.00 0.25 0.40Neg Neg CkJHKIYUJ22/02 1:10 8.84 69.54 1.75 0.00 33.12 H5 H1/3 & Pos APos 1:100 14.32 10.21 0.46 0.14 52.91 H5 & A Pos Pos 1:1,000 12.55 1.410.21 0.15 40.61 H5 & A Pos Pos 1:10.000 11.75 0.00 0.11 −0.07 7.99 A PosNeg 1:100,000 12.86 0.13 0.10 0.00 1.02 A Pos Neg Gs/GYJ337/2006 1:107.88 1.31 0.41 0.19 49.15 H5 & A Pos Pos 1:100 10.06 0.50 0.50 0.3864.63 A Pos Pos 1:1,000 11.89 0.31 0.22 0.21 26.51 A Pos Pos 1:10,00010.79 0.22 0.37 0.16 3.68 A Pos Neg 1:100,000 12.27 0.35 0.57 0.29 0.94Neg IDN/54212006 1:10 7.35 35.64 0.99 0.01 31.02 H5 & A Pos Pos 1:1009.29 6.31 0.30 0.08 49.08 H5 & A Pos Pos 1:1,000 15.86 0.55 0.35 0.4354.28 A Pos Pos 1:10,000 13.83 0.30 0.40 0.27 16.64 A Pos Wk Pos1:100,000 10.77 −0.05 0.34 0.25 4.06 A Pos Neg DkNNM/283/2005 1:10 7.5827.41 0.46 0.16 50.55 H5 &A Pos Pos 1:100 5.46 3.64 0.47 0.24 58.40 H5 &A Pos Pos 1:1,000 8.33 1.13 0.94 0.55 28.40 H5 & A Pos Pos 1:10,000 7.040.34 0.48 0.30 6.03 A Pos Neg 1:100,000 8.75 0.29 0.36 0.18 1.66 A PosNeg

TABLE 5 Mean Results from a Diluition Fluorscent marketed POC HA unitsFactor Units S/N Ratio Test product 10 0 21199.8 392 + 5 2 NT* N/A +/−2.5 4 NT N/A Neg 1.25 8 10488.4 194 NT 1 10 NT N/A Neg 0.5 20 3621.8 67Neg 0.1 100 1087.4 20 NT 0.05 200 532.5 10 NT 0.025 400 354.4 7 NT0.0125 800 86.8 2 NT 0.00625 1600 90.3 2 NT No Virus or N N/A 54.0 N/A*NT = Not Tested, Note Used A/Texas/1/77

Example 5 Influenza Specificity

To test assay specificity, 40 known influenza negative samples weretested. Table 6 shows the results of this particular study. Thedetection specificity of 92% and 97.4% was observed.

TABLE 6 Sample No. Prior Art Run 1 Run 2 1 Neg Neg Neg 2 Neg Neg Neg 3Neg Neg Neg 4 Neg Neg Neg 5 Neg Neg Neg 6 Neg Neg Neg 7 Neg Neg Neg 8Neg Neg Neg 9 Neg Inf. A Inf. A Pos Pos 10 Neg Neg Neg 11 Neg Neg Neg 12Neg Neg Neg 13 Neg Neg Neg 14 Neg Neg Inf. B Pas 15 Neg Neg Neg 16 NegNeg Neg 17 Neg Neg Neg 18 Neg Neg Neg 19 Neg Neg Neg 20 Neg Neg Neg 21Neg Neg Neg 22 Neg Neg Neg 23 Neg Inf. A Inf. A Pos Pos 24 Neg Neg Neg25 Neg Neg Neg 26 Neg Neg Neg 27 Neg Neg Neg 28 Neg Neg Neg 29 Neg NegNeg 30 Neg Neg Neg 31 Neg Neg Neg 32 Neg Neg Neg 33 Neg Neg Neg 34 NegNeg Neg 35 Neg Neg Neg 36 Neg Neg Neg 37 Neg Neg Neg 38 Neg Neg Neg 39Neg Neg Neg 40 Neg Neg Neg

Example 6 NT-proBNP Detection

Sample comprised recombinant peptide in equine serum (40 μl sample+10 μlreagent). Reagent comprised Europium-conjugated mAb-pRNA 6a6. BSA-pRNA(6b6, supra) was striped on a nitrocellulose membrane dipstick testdevice (e.g., FIG. 6). In FIG. 6, lanes 1-4 correspond to 0, 10, 100,1000 peptide at pglmL; reference no. 5 (FIG. 6A) corresponds todetectable NT-proBNP at 10, 100 and 1000 pglmL. FIG. 6 demonstrates thesensitivity using a standard curve calibrated using two standards/levelto obtain fitted concentration of pg/ml, as illustrated in the standardcurve provided in FIG. 9. Based on the calibration data the limit ofdetection was at 1.7 attamoles (10⁻¹⁸) or 0.36 pg/mL NT-proBNP.Furthermore, the limit of quanti tation was about 5.8 attamoles or 1.23pg/mL NT-proBNP. Therefore, the systems of the invention can also beused to quantify an analyte and are quite sensitive. The assay wascalibrated using two standards/level; algorithm=5PL weighted.

Peptide Signal (peak Fitted concentration height) Average concentrationaverage Standard Pg/ml RFU RFU % CV Pg/ml Pg/ml 1 0 227 222 225 1.6 0.00.0 0.0 2 10 3381 3449 3415 1.4 9.9 10.1 10.1 3 50 14792 14409 14601 1.950.7 49.1 49.9 4 125 30408 29976 30192 1.0 126.0 123.5 124.8 5 250 4747745495 46486 3.0 265.1 243.6 254.4 6 500 60915 60166 60540 0.9 494.8475.9 485.4 7 1000 73035 70788 71912 2.2 1137.5 931.2 1034.4 Fittedconcentration, Signal, RFU Pg/ml 343 0.36 (limit of detection =concentration corresponding o average signal of 20 reps of 0 + 3SD) 4000.53 600 1.14 629 1.23 (limit of quantitation = concentrationcorresponding to average signal of 20 reps of 0 + 10SD 1000  2.37

CONCLUSION

The foregoing examples and data demonstrate that the SCD and Test Deviceassay system efficiently and effectively identify Type A and Type Binfluenza strains and are capable of correctly identifying HI. H3, andH5 subtypes. The assay system demonstrated superior performance incomparison to prior art products as measured in terms of influenzadetection specificity and sensitivity—As shown in Table 2, the system'sspecificity for influenza virus was unequivocally demonstrated byshowing no false positive signals toward non-influenza virus orbacterial samples. In addition, as shown in table 5, the specificity forinfluenza detection was 97-4%. In terms of detection sensitivity, testresults shown in Table I and Table 4 demonstrates detection ability indiluted concentration ranges beyond the capabilities of known prior artsystems. For example, H3N2 A/NewYork/5512004 influenza A has tissueculture infectious dose (TCID) 50 titer 3.5 at 1:100 dilution (e.g.,TCID50 3.5 is 5×103 infectious virus particles/mL) for the nucleoproteinantigen (A). The detection sensitivity was seen at the 1:10,000 dilutionwhere the signal to cutoff was seen to be 1.88 for a TCID₅₀ titer ofapproximately 10 TCID50 units/mL for the nucleoprotein antigen (A). Asimilar end point titer was seen with B/Ohio/lS/2001 for thenucleoprotein antigen (B). Furthermore, systems of the invention werecapable of detecting viral concentrations of from between about TCID₅₀10 and about 700, as demonstrated in Table 1. In addition, virus usedfor testing in Table 6 ranged from TCID 6.7 to 9.1 and the detectionsensitivity for these virus ranges from 0.0067 to 0.0097.

1.-18. (canceled)
 19. A method of assaying a sample to detect one ormore analytes comprising: a) providing a sample to a sample collectiondevice (SCD) comprising; i) at least a pair of probes comprising adetection probe and a capture probe, each of which is capable ofspecifically binding an analyte; and ii) an extraction reagent; b)administering a sample from said SCD to a test device (TD) configured todetect said one or more analytes, wherein said TD comprises; i) anaperture for receiving said SCD and where said TD does not comprise amobilizable entity capable of specifically binding said one or moreanalytes; ii) at least one immobilized capture moiety which is capableof specifically binding a component of said capture probe; and therebyassaying said sample to detect said one or more analytes.
 20. The methodof claim 19, wherein said extraction reagent comprises a salt at about0.75M to about 1.125M in a buffered solution.
 21. The method of claim19, wherein said extraction reagent comprises saponin at about 1.0% toabout 1.5% in a buffered solution.
 22. The method of claim 19, whereinsaid extraction reagent contains a zwitterionic agent at about 0.25% toabout 0.5% in a buffered solution.
 23. The method of claim 19, whereinsaid zwitterionic agent is zwittergen 3/17.
 24. The method of claim 19,wherein said one or more analytes comprises one or more virus or viruscomponents.
 25. The method of claim 19, wherein said one or moreanalytes comprises one or more bacteria or bacterial components.
 26. Themethod of claim 19, wherein said one or more analytes comprises one ormore cancer cell or cancer cell component.
 27. The method of claim 19,wherein said one or more analytes comprise one or more analytesassociated with heart damage, heart disease or heart condition.
 28. Themethod of claim 19, wherein said one or more analytes comprises one ormore analytes associated with brain damage, brain disease or braincondition.
 29. The method of claim 24, wherein said virus is influenza Aand/or influenza B.
 30. The method of claim 24, wherein said dropper capcomprises a plurality of pairs of detection and capture probes.
 31. Themethod of claim 29, wherein said influenza A includes subtypes of aformula HxNy, where x is 1 through 16, and y is 1 through 9, or anycombination of xy thereof.
 32. The method of claim 29, wherein saidinfluenza A is H5N1.
 33. The method of claim 19, configured to provide alimit of sensitivity and specificity for detection of said one or moreanalytes of at least about 97%.
 34. The method of claim 19, configuredto provide a limit of sensitivity for detection of said one or moreanalytes of at least about 0.030 pg/ml
 35. The method of claim 19,configured to provide a limit of sensitivity for detection of said oneor more analytes of at least about 1.5 attamoles.
 36. The method ofclaim 24, configured to provide a limit of sensitivity for detection ofsaid one or more different analytes of at least about 10 TCID₅₀ per mLas measured by tissue culture infectious dose 50 (TCID₅₀). 37.-64.(canceled)